Processes using amino acid dehydrogenases and ketoreductase-based cofactor regenerating system

ABSTRACT

The present disclosure relates to the use of an amino acid dehydrogenase in combination with a cofactor regenerating system comprising a ketoreductase. In particular embodiments, the process can be used to prepare L-tert-leucine using a leucine dehydrogenase.

The present application is a Continuation of U.S. patent applicationSer. No. 16/221,223, filed Dec. 14, 2018, now U.S. Pat. No. 10,604,781,which is a Continuation of U.S. patent application Ser. No. 15/627,652,filed Jun. 20, 2017, now U.S. Pat. No. 10,196,667, which is a Divisionalof U.S. patent application Ser. No. 15/184,242, filed Jun. 16, 2016, nowU.S. Pat. No. 9,714,439, which is a Divisional of U.S. patentapplication Ser. No. 14/742,215, filed Jun. 17, 2015, now U.S. Pat. No.9,394,551, which is Divisional of U.S. patent application Ser. No.13/577,772, filed Oct. 16, 2012, now U.S. Pat. No. 9,080,192, which is anational stage application filed under 35 USC § 371 and claims priorityof the international application PCT/US2011/024102, filed Feb. 8, 2011,and U.S. provisional patent application 61/303,179, filed Feb. 10, 2010,each of which is hereby incorporated by reference herein.

TECHNICAL FIELD

The present disclosure relates to biocatalysts and processes forpreparing chiral amino acids using the biocatalysts.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CX2-038USP1_ST25.txt”, a creation date of Feb. 9, 2010,and a size of 48 kilobytes. The sequence listing filed via EFS-Web ispart of the specification and is hereby incorporated in its entirety byreference herein.

BACKGROUND

Amino acid dehydrogenases comprise a group of coenzyme-dependent enzymesthat catalyze the reversible oxidative deamination of an amino acid toits keto acid and ammonia with the concomitant reduction of eithercofactor NAD+, NADP+ or FAD. The enzyme with dehydrogenase properties isdistributed in a number of diverse prokaryotic and eukaryotic organisms.Amino acid dehydrogenases have been studied widely because of theirpotential applications in biosensors or diagnostic kits, synthesis of L-and D-amino acids for use in production pharmaceutical peptides,herbicides and insecticides (Brunhuber et al., 1994, Crit. Rev. Biochem.Mol. Biol. 29(6):415-467; Hummel et al., 1989, Eur. J. Biochem.184:1-13; Krix et al., 1997, J. Biotech. 53:29-39; Ohshima et al., 1990,Adv. Biochem. Eng. Biotechnol. 42:181-209; U.S. Pat. No. 7,550,277). Forexample, the anti-hypertensives ramipril, enalapril, benazapril, andprinivil are prepared using L-homophenylalanine, and certain secondgeneration pril analogs are synthesized from p-substituted-Lhomophenylalanine. Certain β-lactam antibiotics use substitutedD-phenylglycine side chains, and while other antibiotics are based onaminoadipic acid and other unnatural amino acids. The unnatural aminoacids L-tert-leucine, L-nor-valine, L-nor-leucine,L-2-amino-5-[1,3]dioxolan-2yl-pentanoic acid have been used as aprecursor in the synthesis of a number of different developmental drugs.The enzyme leucine dehydrogenase and mutants thereof have been shown tobe capable of catalyzing the reductive amination of the corresponding2-ketoacids of alkyl and branched-chain amino acids, and L-tert-leucinehas been produced commercially with such an enzyme.

Given the industrial utility of L- and D-amino acid dehydrogenases, itis desirable to develop processes and systems that can enhance thebiocatalytic reactions carried out by amino acid dehydrogenases.

SUMMARY

The present disclosure provides coupled systems for the efficientbiosynthesis of chiral amino acid compounds using an L- or D-amino aciddehydrogenase (“AADH”) coupled with a cofactor regenerating systemcomprising a ketoreductase (“KRED”).

In certain embodiments, the present disclosure provides a process forconverting a 2-oxo acid compound of formula I which is a substrate foran amino acid dehydrogenase to a chiral amino acid of formula IIa:

comprising contacting the compound of formula I with a reaction mediumcomprising an amino acid dehydrogenase, an ammonium ion donor, NAD+/NADHor NADP+/NADPH, and a cofactor regenerating system comprising aketoreductase and a lower secondary alcohol, under conditions where thecompound of formula I is converted to the chiral amino acid of formulaIIa and the lower secondary alcohol is converted to a ketone. In certainembodiments of the process, R is a substituted or unsubstituted(C₁-C₁₀)alkyl, —(C₂-C₆)alkenyl, —(C₂-C₆)alkynyl, —(C₃-C₈)cycloalkyl,heterocycloalkyl, aryl, or heteroaryl. In certain embodiments, R is asubstituted or unsubstituted group selected from those groups shown ineither of Table 1 or Table 2 disclosed herein.

In certain embodiments of the process for converting a compound offormula I which is a substrate for an amino acid dehydrogenase to achiral amino acid of formula IIa, the amino acid dehydrogenase is anL-amino acid dehydrogenase and the chiral amino acid of formula IIa isIIb,

wherein the product of formula IIb is formed in enantiomeric excess. Incertain embodiments, the L-amino acid dehydrogenase is from Bacillus,Clostridium, Corynebacterium, Geobacillus, Natronobacterium,Synechocystis, Thermoactinomyces, Thermos, Thermomicrobium, or Carderia.In some embodiments, the L-amino acid dehydrogenase is selected fromL-alanine dehydrogenase, L-aspartate dehydrogenase,L-erythro-3,5-diaminohexanoate dehydrogenase, L-leucine dehydrogenase,L-glutamate dehydrogenase, lysine dehydrogenase, L-phenylalaninedehydrogenase, L-serine dehydrogenase, L-valine dehydrogenase,L-2,4-diaminopentanoate dehydrogenase, L-glutamate synthase,L-diaminopimelate dehydrogenase, L-N-methylalanine dehydrogenase,L-lysine 6-dehydrogenase, and L-tryptophan dehydrogenase.

In certain embodiments of the process for converting a compound offormula I which is a substrate for an amino acid dehydrogenase to achiral amino acid of formula IIa, the amino acid dehydrogenase is aD-amino acid dehydrogenase and the chiral amino acid of formula IIa isIIc,

wherein the compound of formula IIc is formed in enantiomeric excess. Incertain embodiments of the process, the D-amino acid dehydrogenase isfrom Halobacterium, Methanosarcina, Pseudomonas, Pyrobaculum,Salmonella, Corynebacterium, and Escherichia. In certain embodiments,the D-amino acid dehydrogenase is selected from a D-alaninedehydrogenase, D-threonine dehydrogenase, and D-proline dehydrogenase.

In certain embodiments of the process for converting a compound offormula I which is a substrate for an amino acid dehydrogenase to achiral amino acid of formula IIa, the amino acid dehydrogenase comprisesa L-leucine dehydrogenase, the compound of formula I is3,3-dimethyl-2-oxobutanoic acid, the product of formula IIa is(S)-2-amino-3,3-dimethylbutanoic acid. In some embodiments of theprocess, the leucine dehydrogenase is a wild type leucine dehydrogenaseor an engineered leucine dehydrogenase. In some embodiments, the leucinedehydrogenase is from Bacillus, Clostridium, Corynebacterium,Geobacillus, Natronobacterium, Thermoactinomyces, Thermos,Thermomicrobium, or Carderia. In some embodiments, the leucinedehydrogenase is from Bacillus acidokaludarius, Bacillus brevis,Bacillus caldolyticus, Bacillus cereus, Bacillus megaterium, Bacillusmesentericus, Bacillus mycoides, Bacillus natto, Bacillus pumilus,Bacillus sp., Bacillus sphaericus, Bacillus stearothermophilus, Bacillussubtilis, Clostridium thermoaceticum, Corynebacteriumpseudodiphtheriticum, Geobacillus stearothermophilus, Natronobacteriummagadii, or Thermoactinomyces intermedius. In some embodiments, theleucine dehydrogenase comprises the amino acid sequence of SEQ ID NO:26.

In certain embodiments, the present disclosure provides a process forproducing (S)-2-amino-3,3-dimethylbutanoic acid, comprising: contacting3,3-dimethyl-2-oxobutanoic acid with a leucine dehydrogenase in areaction medium comprising an ammonium ion donor, cofactor NAD+/NADH orNADP+/NADPH, and a cofactor recycling system comprising a ketoreductaseand a lower secondary alcohol, under conditions where the3,3-dimethyl-2-oxobutanoic acid is converted to(S)-2-amino-3,3-dimethylbutanoic acid, wherein the3,3-dimethyl-2-oxobutanoic acid is at about 75 g/L to 125 g/L, thecofactor is at about 0.30 g/L to 0.70 g/L, and the leucine dehydrogenaseand ketoreductase are each independently at about 0.5 to about 1.0 g/L.In certain embodiments of the process, the secondary alcohol is presentin at least 1.5 fold stoichiometric excess of substrate. In someembodiments of the process, the secondary alcohol is isopropanol,wherein the isopropanol is at about 7% to 12% volume of the reactionmedium by (weight/volume).

In certain embodiments, the present disclosure provides a process forconverting a compound mixture of formula IId which comprises a substratefor an amino acid dehydrogenase to a composition of formula I and achiral amino acid of formula IIa:

where the process comprises contacting the compound mixture of formulaIId with an enantioselective amino acid dehydrogenase in a reactionmedium comprising NAD+/NADH or NADP+/NADPH and a cofactor recyclingsystem comprising a ketoreductase and a lower alkyl ketone, underconditions where the compound mixture of formula IId is converted to thecomposition of formula I and a chiral amino acid of formula IIa, and thelower alkyl ketone is converted to a lower secondary alcohol. In someembodiments of the process, the compound mixture of IId is a racemicmixture of formula IIe:

In some embodiments of the process, the amino acid dehydrogenasecomprises an L-amino acid dehydrogenase and the chiral amino acid offormula IIa is IIc

wherein the process results in chiral amino acid of formula IIc inenantiomeric excess. In some embodiments of the process, the amino aciddehydrogenase comprises a D-amino acid dehydrogenase and the chiralamino acid of formula IIa is IIb

wherein the process results in a chiral amino acid of formula IIb inenantiomeric excess.

In certain embodiments, the present disclosure provides a process forpreparing an N-protected amino acid compound, wherein the methodcomprises: (i) contacting a compound of formula I with a reaction mediumcomprising an amino acid dehydrogenase, an ammonium ion donor, NAD+/NADHor NADP+/NADPH, and a cofactor regenerating system comprising aketoreductase and a lower secondary alcohol under suitable conditionswhere the compound of formula I is converted to the chiral amino acidcompound of formula IIa and the lower secondary alcohol is converted toa ketone; and (ii) contacting the amino acid compound of formula IIawith a compound comprising an N-protecting group under conditions, wherethe N-protecting group reacts with the compound of formula IIa to forman N-protected amino acid compound.

In certain embodiments of the process for preparing an N-protected aminoacid compound, the biocatalytic step comprises contacting3,3-dimethyl-2-oxobutanoic acid with a leucine dehydrogenase in areaction medium comprising an ammonium ion donor, cofactor NAD+/NADH orNADP+/NADPH, and a cofactor recycling system comprising a ketoreductaseand a lower secondary alcohol, under conditions where the3,3-dimethyl-2-oxobutanoic acid is converted to(S)-2-amino-3,3-dimethylbutanoic acid. In some embodiments, the3,3-dimethyl-2-oxobutanoic acid is at about 75 g/L to 125 g/L, thecofactor is at about 0.30 g/L to 0.70 g/L, and the leucine dehydrogenaseand ketoreductase are each independently at about 0.5 to about 1.0 g/L.In some embodiments, the secondary alcohol is present in at least 1.5fold stoichiometric excess of substrate. In some embodiments, thesecondary alcohol is isopropanol, and the isopropanol is at about 7% to12% volume of the reaction medium by (weight/volume). In someembodiments, the N-protecting group is selected from Cbz, FMOC, BOC andMOC.

In certain embodiments of the various processes for preparing chiralamino acid compounds disclosed herein, the process is carried out in acell free system. In some embodiments of the various processes, theamino acid dehydrogenase is present as a crude extract, and in someembodiments, the amino acid dehydrogenase is substantially purified.

In certain embodiments of the various processes for preparing chiralamino acid compounds disclosed herein, the ketoreductase is a wild typeketoreductase or an engineered ketoreductase. In some embodiments, theketoreductase is from Lactobacillus, Candida, Novosphingobium, orSaccharomyces, and in some embodiments, the ketoreductase is from anorganism selected from Lactobacillus kefir, Lactobacillus brevis,Lactobacillus minor, Candida magnoliae, Saccharomyces cerevisiae, andNovosphingobium aromaticivorans. In some embodiments, the ketoreductaseis an engineered ketoreductase derived from the wild-type ketoreductaseof Novosphingobium aromaticivorans, wherein the engineered ketoreductasecomprising an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, 99%, or more identity to a sequence selected from SEQ ID NO: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, and 24.

In certain embodiments of the various processes for preparing chiralamino acid compounds disclosed herein, the ketoreductase ischaracterized by increased thermostability, increased solvent stability,and/or increased enzymatic activity relative to a referenceketoreductase. In some embodiments, the ketoreductase used in thecofactor recycling system has an improved property over a referenceketoreductase of increased activity in the conversion of the lowersecondary alcohol (e.g., isopropanol) of the recycling system to thecorresponding lower alkyl ketone. In some embodiments, the ketoreductasehaving the increased activity in the conversion of the lower secondaryalcohol is at least 2.0 fold, 2.5 fold, 5.0 fold, 7.5 fold, 10-fold, ormore improved relative to a reference ketoreductase (e.g., a referenceketoreductase of SEQ ID NO: 2).

In certain embodiments of the various processes for preparing chiralamino acid compounds disclosed herein, the ketoreductase used in thecofactor recycling system has an improved property over a referenceketoreductase of decreased or no activity with the compound of formula I(e.g., trimethylpyruvic acid) which is a substrate for the amino aciddehydrogenase used in the process. In some embodiments of the process,the activity of the ketoreductase used in the cofactor recycling systemwith the compound of formula I is less than about 5%, 2%, 1%, 0.5%,0.1%, 0.05%, 0.01%, or an even smaller %, of the activity of the aminoacid dehydrogenase used in the process with the compound of formula I.In some embodiments of the process, the ketoreductase used in thecofactor recycling system has no activity with the compound of formulaI.

In certain embodiments of the various processes for preparing chiralamino acid compounds disclosed herein, the ketoreductase used in thecofactor recycling system is an engineered ketoreductase is capable ofrecycling cofactor by converting isopropanol (IPA) to acetone in areaction medium of 3 to 20% IPA at a pH of about 9.0 to 10.5 with anactivity at least 1.5-fold greater than the reference ketoreductase ofSEQ ID NO: 2.

In certain embodiments of the various processes for preparing chiralamino acid compounds disclosed herein, the process further comprisesremoving from the reaction medium the ketone formed from the lowersecondary alcohol, and in certain embodiments the lower secondaryalcohol is isopropanol and the ketone removed is acetone. In someembodiments, the secondary alcohol is present in at least 1.5 foldstoichiometric excess of substrate.

In certain embodiments of the various processes for preparing chiralamino acid compounds disclosed herein, the reaction medium is at a pH ofabout 8.5 to about 10.5, or a pH of about 8.5 to about 9.5, or a pH ofabout 9.0.

In certain embodiments of the various processes for preparing chiralamino acid compounds disclosed herein, the reaction medium is at atemperature of about 25° C. to about 45° C., or about 35° C. to about40° C.

DETAILED DESCRIPTION Definitions

As used herein, the following terms are intended to have the followingmeanings.

“Protein,” “polypeptide,” “oligopeptide,” and “peptide” are usedinterchangeably to denote a polymer of at least two amino acidscovalently linked by an amide bond, regardless of length orpost-translational modification (e.g., glycosylation, phosphorylation,lipidation, myristilation, ubiquitination, etc.). Included within thisdefinition are D- and L-amino acids, and mixtures of D- and L-aminoacids.

“Amino acid dehydrogenase” or “AADH” are used interchangeably herein torefer to a polypeptide capable of carrying out the conversion of anamino acid, in the presence of an electron acceptor, to a 2-oxo acid,NH₃, and reduced acceptor. In some embodiments, amino aciddehydrogenases are also capable of carrying out the reverse reaction ofconverting the 2-oxo acid, in the presence of an ammonium ion donor andan electron donor, to an amino acid and oxidized electron donor. L-aminoacid dehydrogenase refers to an amino acid dehydrogenase that isstereospecific or stereoselective for an L-amino acid. D-amino aciddehydrogenase refers to an amino acid dehydrogenase that isstereospecific or stereoselective for a D-amino acid. Generally, theelectron acceptor/donor for the amino acid dehydrogenase is nicotinamideadenine dinucleotide in oxidized/reduced form (i.e., NAD+/NADH) ornicotinamide adenine dinucleotide phosphate in oxidized/reduced form(i.e., NADP+/NADPH). Amino acid dehydrogenase as used herein includenaturally occurring (wild type) amino acid dehydrogenases as well asnon-naturally occurring polypeptides generated by human manipulation(e.g., recombinant or engineered enzymes).

“Ketoreductase” and “KRED” are used interchangeably herein to refer to apolypeptide that is capable of reducing a ketone to an alcohol productand/or oxidizing an alcohol to a ketone product. The polypeptidetypically utilizes a cofactor reduced nicotinamide adenine dinucleotide(NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) asthe reducing agent; or may use the corresponding NAD+ or NADPH+ asoxidizing agent. Ketoreductases as used herein include naturallyoccurring (wild type) ketoreductases as well as non-naturally occurringpolypeptides generated by human manipulation (e.g., recombinant orengineered enzymes). They may be used to effect one or more chemicaltransformations, including the regeneration of cofactors such as NAD(P)Hor NAD(P)⁺.

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Recombinant” or “engineered” or “non-naturally occurring” when usedwith reference to, e.g., a cell, nucleic acid, or polypeptide, refers toa material, or a material corresponding to the natural or native form ofthe material, that has been modified in a manner that would nototherwise exist in nature, or is identical thereto but produced orderived from synthetic materials and/or by manipulation usingrecombinant techniques (e.g., genetic engineering). Non-limitingexamples include, among others, recombinant cells expressing genes thatare not found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise expressed at a different level.

“Derived from” identifies the originating polypeptide, and/or the geneencoding such polypeptide, upon which the engineering was based.

“Substrate” refers to a substance or compound that is converted or meantto be converted into another compound by the action of an enzyme. Theterm includes aromatic and aliphatic compounds, and includes not only asingle compound, but also combinations of compounds, such as solutions,mixtures and other materials which contain at least one substrate.

“Stereoselectivity” refers to the preferential formation in a chemicalor enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly reported in the art (typically as a percentage) asthe enantiomeric excess calculated therefrom according to the formula[major enantiomer−minor enantiomer]/[major enantiomer+minor enantiomer].Where the stereoisomers are diastereoisomers, the stereoselectivity isreferred to as diastereoselectivity, the fraction (typically reported asa percentage) of one diastereomer in the sum with others.

“Stereospecific” refers to the preferential conversion in a chemical orenzymatic reaction of one stereoisomer over another. Stereospecificitycan be partial, where the conversion of one stereoisomer is favored overthe other, or it may be complete where only one stereoisomer isconverted.

“Increased enzymatic activity” refers to an improved property of anenzyme, which can be represented by an increase in specific activity(e.g., product produced/time/weight protein) or an increase in percentconversion of the substrate to the product (e.g., percent conversion ofstarting amount of substrate to product in a specified time period usinga specified amount of KRED) as compared to a reference enzyme. Exemplarymethods to determine enzyme activity are provided in the Examples. Anyproperty relating to enzyme activity may be affected, including theclassical enzyme properties of Km, Vmax or kcat, changes of which canlead to increased enzymatic activity. Improvements in enzyme activitycan be from about 1.5 times the enzymatic activity of the correspondingwild-type ketoreductase enzyme, to as much as 2 times. 5 times, 10times, 20 times, 25 times, 50 times, 75 times, 100 times, or moreenzymatic activity than the naturally occurring ketoreductase or anotherengineered ketoreductase from which the ketoreductase polypeptides werederived. In specific embodiments, the engineered enzyme exhibitsimproved enzymatic activity in the range of 1.5 to 50 times, 1.5 to 100times greater than that of the parent enzyme. It is understood by theskilled artisan that the activity of any enzyme is diffusion limitedsuch that the catalytic turnover rate cannot exceed the diffusion rateof the substrate, including any required cofactors. The theoreticalmaximum of the diffusion limit, or kcat/Km, is generally about 10⁸ to10⁹ (M-1 s-1). Hence, any improvements in the enzyme activity of anenzyme will have an upper limit related to the diffusion rate of thesubstrates acted on by the enzyme. Comparisons of enzyme activities aremade using a defined preparation of enzyme, a defined assay under a setcondition, and one or more defined substrates, as further described indetail herein. Generally, when lysates are compared, the numbers ofcells and the amount of protein assayed are determined as well as use ofidentical expression systems and identical host cells to minimizevariations in amount of enzyme produced by the host cells and present inthe lysates.

“Conversion” refers to the enzymatic transformation of the substrate tothe corresponding product. “Percent conversion” refers to the percent ofthe substrate that is converted to the product within a period of timeunder specified conditions. Thus, the “enzymatic activity” or “activity”of an enzyme(s) can be expressed as “percent conversion” of thesubstrate to the product.

“Improved thermostability” and “improved thermal stability” are usedinterchangeably herein to refer to a property of increased resistance toinactivation when exposed to a set temperature or set of temperatures indefined conditions as compared to the resistance to inactivation of areference enzyme. Activity of the enzyme pre- and post treatment aremeasured under the same defined assay condition. Thermostability canalso be compared and expressed as the temperature at which half of theinitial activity is retained after a defined incubation time after anincrease from one temperature to another, i.e., from X° C. to Y° C.“Residual activity” or “residual enzyme activity” refers to the activitythat remains following exposure to the set temperature in a definedcondition.

“Solvent stable” refers to a polypeptide that maintains similar activity(more than e.g., 60% to 80%) after exposure to varying concentrations(e.g., 5-99%) of solvent (isopropyl alcohol, tetrahydrofuran,2-methyltetrahydrofuran, acetone, toluene, butylacetate, methyltert-butylether, etc.) for a period of time (e.g., 0.5-24 hrs) comparedto the untreated polypeptide.

“pH stable” refers to a polypeptide that maintains similar activity(more than e.g., 60% to 80%) after exposure to high or low pH (e.g.,4.5-6 or 8 to 12) for a period of time (e.g., 0.5-24 hrs) compared tothe untreated polypeptide.

“Thermo- and solvent stable” refers to a polypeptide that is boththermostable and solvent stable, particularly with respect to itsbiological function, e.g., enzymatic activity.

“Cofactor” refers a substance that is necessary or beneficial to theactivity of an enzyme. In the context of an amino acid dehydrogenase,the cofactor is generally a nicotinamide cofactor. “Nicotinamidecofactor” refers to any type of the oxidized and reduced forms ofnicotinamide adenine dinucleotide (NAD+ and NADH, respectively) and theoxidized and reduced forms of nicotinamide adenine dinucleotidephosphate (NADP+ and NADPH, respectively) and derivatives and analogsthereof. With regard to a nicotinamide cofactor, the term “derivative”means any compound containing a pyridine structural element, includingnicotinamides that have been chemically modified by attachment tosoluble or insoluble polymeric materials. Some examples of derivativesof nicotinamide cofactors are described in U.S. Pat. No. 5,106,740, andMansson and Mosbach, 1987, Methods in Enzymology 136:3 45, thedisclosures of which are incorporated herein by reference. The term“analogs,” as used herein, refers to materials that undergo a formalhydride transfer in a redox reaction similar to that undergone bynicotinamide cofactors. Examples of analogs of nicotinamide cofactorsuseful in the practice of the present process include compoundsdescribed in U.S. Pat. No. 5,801,006, the disclosure of which isincorporated herein by reference. Other suitable cofactors, as definedherein, can be used in the practice of the invention, as would berecognized by those skilled in the art.

“Cofactor regenerating system” and “cofactor recycling system” are usedinterchangeably herein to refer to a set of reactants that participatein a reaction that reduces the oxidized form of the cofactor (e.g.,NADP+ to NADPH). In the embodiments herein, cofactors oxidized by theamino acid dehydrogenase-catalyzed reaction are regenerated in reducedform by the cofactor regenerating system. Cofactor regenerating systemscomprise a stoichiometric reductant that is a source of reducinghydrogen equivalents and is capable of reducing the oxidized form of thecofactor. The cofactor regenerating system may further comprise acatalyst, for example an enzyme catalyst, that catalyzes the reductionof the oxidized form of the cofactor by the reductant.

“Alkyl” by itself or as part of another substituent refers to asaturated branched or straight hydrocarbon chain derived by the removalof one hydrogen atom from a single carbon atom of a parent alkane. Alkylgroups include, but are not limited to, methyl; ethyl; propyls, such aspropan-1-yl, propan-2-yl (isopropyl), etc.; butyls, such as butan-1-yl,butan-2-yl (sec-butyl), 2-methyl propan-1-yl (isobutyl), 2-methylpropan-2-yl (t-butyl), etc.; and the like. In some embodiments, thealkyl groups are (C₁-C₆) alkyl. “Lower alkyl” refers to a straight-chainor branched saturated aliphatic hydrocarbon having 1 to 6, preferably 1to 4, carbon atoms. Typical lower alkyl groups include methyl, ethyl,propyl, isopropyl, butyl, t-butyl, 2-butyl, pentyl, hexyl and the like.

“Alkenyl” refers to by itself or as part of another substituent refersto an unsaturated branched, straight chain or cyclic alkyl having atleast one carbon-carbon double bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkene. The groupmay be in either the cis or trans conformation about the double bond(s).Alkenyl groups include, but are not limited to, ethenyl; propenyls suchas prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.;and the like. In some embodiments, the alkenyl group is (C₂-C₆) alkenyl.

“Alkynyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon triple bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkyne. Typical alkynyl groupsinclude, but are not limited to, ethynyl; propynyls such asprop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. In some embodiments,the alkynyl group is (C2-C6) alkynyl.

“Heteroalkyl,” Heteroalkanyl,” Heteroalkenyl,” Heteroalkynyl,”Heteroalkyldiyl” and “Heteroalkyleno” by themselves or as part ofanother substituent refer to alkyl, alkanyl, alkenyl, alkynyl, alkyldiyland alkyleno groups, respectively, in which one or more of the carbonatoms are each independently replaced with the same or differentheteratoms or heteroatomic groups. Heteroatoms and/or heteroatomicgroups which can replace the carbon atoms include, but are not limitedto, —O—, —S—, —S—O—, —NR′—, —PH—, —S(O)—, —S(O)₂—, —S(O) NR′—,—S(O)₂NR′—, and the like, including combinations thereof, where each R′is independently hydrogen or (C₁-C₆) alkyl.

“Cycloalkyl” and “Heterocycloalkyl” by themselves or as part of anothersubstituent refer to cyclic versions of “alkyl” and “heteroalkyl”groups, respectively. For heteroalkyl groups, a heteroatom can occupythe position that is attached to the remainder of the molecule. Typicalcycloalkyl groups include, but are not limited to, cyclopropyl;cyclobutyls such as cyclobutanyl and cyclobutenyl; cyclopentyls such ascyclopentanyl and cyclopentenyl; cyclohexyls such as cyclohexanyl andcyclohexenyl; and the like. Typical heterocycloalkyl groups include, butare not limited to, tetrahydrofuranyl (e.g., tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, etc.), piperidinyl (e.g., piperidin-1-yl,piperidin-2-yl, etc.), morpholinyl (e.g., morpholin-3-yl,morpholin-4-yl, etc.), piperazinyl (e.g., piperazin-1-yl,piperazin-2-yl, etc.), and the like.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon group having the stated number of carbonatoms (i.e., C₅-C₁₅ means from 5 to 15 carbon atoms) derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Aryl groups include, but are not limited to,groups derived from aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, coronene, fluoranthene,fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene,indane, indene, naphthalene, octacene, octaphene, octalene, ovalene,penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene,phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene,triphenylene, trinaphthalene, and the like, as well as the various hydroisomers thereof. In some embodiments, the aryl group is (C₅-C₁₅) aryl,with (C₅-C₁₀) being even more preferred. In some embodiments, the arylsare cyclopentadienyl, phenyl and naphthyl.

“Heteroaryl” by itself or as part of another substituent refers to amonovalent heteroaromatic group having the stated number of ring atoms(e.g., “5-14 membered” means from 5 to 14 ring atoms) derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, benzimidazole,benzisoxazole, benzodioxan, benzodiaxole, benzofuran, benzopyrone,benzothiadiazole, benzothiazole, benzotriazole, benzoxazine,benzoxazole, benzoxazoline, carbazole, 3-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike, as well as the various hydro isomers thereof. In some embodiments,the heteroaryl group is a 5-14 membered heteroaryl. In some embodiments,the heteroaryl group is a 5-10 membered heteroaryl.

“Substituted” when used to modify a specified group or radical, meansthat one or more hydrogen atoms of the specified group or radical areeach, independently of one another, replaced with the same or differentsubstituent(s). Each substituent can be the same or different. Examplesof suitable substituents include, but are not limited to, alkyl,alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, cycloheteroalkyl,heteroaryl, OR^(a) (e.g., hydroxyl, alkoxy (e.g., methoxy, ethoxy, andpropoxy), aryloxy, heteroaryloxy, aralkyloxy, ether, ester, carbamate,etc.), hydroxyalkyl, alkoxycarbonyl, alkoxyalkoxy, perhaloalkyl,perfluoroalkyl (e.g., CF₃, CF₂, CF₃), perfluoroalkoxy (e.g., OCF₃,OCF₂CF₃), alkoxyalkyl, SR^(a) (e.g., thiol, alkylthio, arylthio,heteroarylthio, aralkylthio, etc.), S⁺R^(a) ₂, S(O)R^(a), SO₂R^(a),NR^(a)R^(a) (e.g., primary amine (i.e., NH₂), secondary amine, tertiaryamine, amide, carbamate, urea, etc.), hydrazide, halide, nitrile, nitro,sulfide, sulfoxide, sulfone, sulfonamide, thiol, carboxy, aldehyde,keto, carboxylic acid, ester, amide, imine, and imide, including selenoand thio derivatives thereof, wherein each of the substituents can beoptionally further substituted. In embodiments in which a functionalgroup with an aromatic carbon ring is substituted, such substitutionswill typically number less than about 10 substitutions, more preferablyabout 1 to 5, with about 1 or 2 substitutions being preferred.

“Amino acid” refers to a molecule having the general formulaNHR^(b)—CHR^(b′)—COOH (wherein R is H, and R^(b′) is an amino acid sidechain, or R and R^(b′) together with the carbon and nitrogen to whichthey are bonded form a ring, e.g., proline) which is capable of forminga peptide bond with one or more other molecules having the same generalformula. The term embraces both L and D amino acids. A “chiral aminoacid” refers to an amino acid in which the α-carbon is an asymmetriccarbon atom, which is a carbon atom bonded to four different entities,such that an interchanging of any two groups gives rise to anenantiomer. In the context of an amino acid, a chiral amino acid ofgeneral formula NHR—CHR′—COOH, the R′ group is an amino acid side chainother than H. A chiral amino acid can be an L-amino acid or a D-aminoacid.

“Alcohol” refers to an alkyl group in which one or more of the hydrogenatoms has been replaced by an —OH group. A “lower alcohol” refers to analcohol in which the alkyl group is about 1 to about 6 carbon atoms.A“secondary alcohol” refers to an alcohol in which the —OH group isbonded to a carbon atom that is bonded to one hydrogen atom and to twoother carbon atoms, such as in 2-propanol (isopropanol), 2-butanol,2-hexanol and the like. A “lower secondary alcohol” refers to asecondary alcohol in which the alkyl group is of 3 to about 6 carbonatoms.

“Ketone” refers to a carbonyl compound of general formula R′—C(O)—R″ inwhich the carbonyl carbon is bonded to two carbon atoms. In someembodiments, R′ and R″ are the same and in some embodiments, R′ and R″are each independently an optionally substituted alkyl or aryl. A loweralkyl ketone refers to a carbonyl compound of general formula R′—C(O)—R″in which R′ and R″ is each an alkyl of C₁ to C₅ carbon atoms, where thetotal number of carbon atoms in the ketone is 3 to 6 carbon atoms.

“Protecting group” refers to a group of atoms that, when attached to areactive functional group in a molecule, mask, reduce or prevent thereactivity of the functional group. Typically, a protecting group may beselectively removed as desired during the course of a synthesis.Examples of protecting groups can be found in P. G. M. Wuts and T. W.Greene, “Greene's Protective Groups in Organic Synthesis—FourthEdition,” John Wiley and Sons, New York, N.Y., 2007, Chapter 7(“Greene”) which chapter is hereby incorporated by reference in itsentirety, and Harrison et al., Compendium of Synthetic Organic Methods,Vols. 1-8, 1971-1996, John Wiley & Sons, NY.

“N-protecting group” (or nitrogen protecting group) means a substituentcommonly employed to block or protect a nitrogen functionality (e.g.,the amine nitrogen of an amino acid) while carrying out a reaction withother functional groups on a compound. Accordingly, an “N-protected”compound refers to a modified form of the compound where an N-protectinggroup is blocking a nitrogen functionality on the compound fromundergoing reaction.

“Ammonium source” or “ammonium ion donor” refers to a compound orcomposition that forms ammonia or ammonium ion in a reaction medium.

“Reaction medium” refers to a solution comprising a mixture of two ormore components (e.g., enzyme, substrate, cofactor) which can undergoreaction in the solution. For the enzymatic reactions described hereinthe reaction medium typically is an at least partially aqueous solution.In some embodiments, the reaction medium comprises aqueous and organicsolvents (e.g., isopropanol), one or more phases.

“Isolated polypeptide” refers to a polypeptide which is separated fromother contaminants that naturally accompany it, e.g., protein, lipids,and polynucleotides. The term embraces polypeptides which have beenremoved or purified from their naturally-occurring environment orexpression system (e.g., host cell or in vitro synthesis).

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure ketoreductase composition willcomprise about 60% or more, about 70% or more, about 80% or more, about90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.In some embodiments, the object species is purified to essentialhomogeneity (i.e., contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), and elemental ion species arenot considered macromolecular species.

-   -   Processes Using Amino Acid Dehydrogenases Coupled To        Ketoreductase-Based Cofactor Regenerating System

The present disclosure provides a process for the conversion of a 2-oxoacid (i.e., a keto acid) to an amino acid in the presence of an ammoniumsource in a reaction mediated by an amino acid dehydrogenase (“AADH”)and a cofactor recycling system comprising a ketoreductase (“KRED”), asgenerally depicted in Scheme 1.

The stereoselectivity of the amino acid dehydrogenase can be exploitedto carry out the reverse reaction, i.e., conversion of an amino acid tothe 2-oxo acid, and permit chiral resolution of L- and D-amino acids.

In the conversion of the 2-oxo acid to the amino acid, amino aciddehydrogenases typically use a cofactor, generally nicotinamide adeninedinucleotide (NAD+/NADH) or nicotinamide adenine dinucleotide phosphate(NADP+/NADPH). To enhance the amino acid dehydrogenase-mediated process,a cofactor regenerating system of formate dehydrogenase, glucosedehydrogenase, or phosphite dehydrogenase have been used to convert theoxidized NAD+/NADP+ to the reduced form NADH/NADPH. See, e.g., US patentpublication 2009087995; US patent publication 20090117627; EP1925674;Johannes et al., 2005, Appl Environ Microbiol. 71(10):5728-5734;Johannes et al., 2007, Biotechnol Bioeng. 96(1):18-26; McLachan et al.,2008, Biotechnol Bioeng. 99(2):268-274). By continual replenishment ofthe reduced NADH or NADPH, the equilibrium of the amino aciddehydrogenase mediated process can be shifted towards product formation,thereby increasing the conversion of the oxo acid to the amino acidproduct. A whole cell-based system for conversion of D-amino acid toL-amino acid using amino oxidase, amino acid dehydrogenase, and acofactor regenerating system is described in U.S. Pat. No. 7,217,544.The patent publication describes the use of formate dehydrogenase,malate dehydrogenase or alcohol dehydrogenase activities present inwhole cells for regeneration of the cofactor. A coupled enzyme system ofa phenylalanine dehydrogenase and a cofactor regenerating system usingan alcohol dehydrogenase is described in Paradisi et al., 2007, J.Biotech. 128:408-411. However, in Paradisi et al., ethanol was employedas the substrate for the alcohol dehydrogenase, thereby formingacetaldehyde as the product.

As provided in the present disclosure, it has been found that processesusing amino acid dehydrogenases when carried out in the presence of acofactor regenerating system comprising a ketoreductase and an alcoholcan be used for the efficient conversion of an oxo acid to itscorresponding amino acid. In particular, use of a lower secondaryalcohol for the ketoreductase-based cofactor regenerating system canincrease the conversion of oxo acid to the amino acid in the amino aciddehydrogenase catalyzed reaction, avoiding the production ofacetaldehyde which can react with and inactivate enzymes. Moreover, theketone product formed by the ketoreductase catalyzed reaction, such asacetone, is less volatile than acetaldehyde, thereby providing greatercontrol over the reaction, particularly in larger scale processes (e.g.,reaction medium of 50 L, 100 L, 300 L, 500 L, or even greater volume).

Further, the loss of process control due to the volatility ofacetaldehyde would create greater difficulty when using an amino aciddehydrogenase in the reverse reaction (e.g., converting amino acid tothe corresponding oxo acid) as described in greater detail herein. Inparticular, pushing the equilibrium of the reverse reaction would befacilitated with a less volatile lower alkyl ketone such as acetone.

Although advantageously less volatile than acetaldehyde, the ketoneproduct formed by the ketoreductase reaction of a lower secondaryalcohol (e.g., acetone) is sufficiently volatile to allow its facileremoval from the reaction medium thereby shifting the equilibrium of theketoreductase mediated process towards further cofactor reduction, andfurther conversion of oxo acid to the amino acid by the amino aciddehydrogenase. Consequently, the combination of a ketoreductase andlower secondary alcohol provides the advantages greater reaction control(and increased safety) and enhanced ability to drive the desired aminoacid dehydrogenase reaction to completion.

Significant benefit can be further obtained when engineeredketoreductases that have improved enzyme properties, including amongothers, increased enzymatic activity, increased thermostability,increased solvent stability and/or increased pH stability are used inthe process. Engineered ketoreductases with such improved properties canallow use of conditions not well tolerated by the naturally occurringenzymes, including conditions such as, for example, high oxo acidconcentration, high alcohol concentration, high ammonium ion donorconcentration, elevated incubation temperatures, and increasedincubation times. Use of engineered ketoreductases also can reduce theamount of enzyme needed in the process.

Accordingly, the present disclosure provides a process for converting a2-oxo acid compound of formula I that is a substrate for an amino aciddehydrogenase to a chiral amino acid compound of formula IIa,

in a reaction medium comprising NAD+/NADH or NADP+/NADPH, and a cofactorregenerating system, where the cofactor regenerating system comprises aketoreductase and a secondary alcohol. In particular, the secondaryalcohol is a lower secondary alcohol, such as isopropanol, 2-butanol,3-methyl-2-butanol, 2-pentanol, 3-pentanol, or 3,3-dimethyl-2-butanol.

In some embodiments, the process comprises contacting the 2-oxo acidcompound of formula I with a reaction medium comprising an amino aciddehydrogenase, an ammonium ion donor, NAD+/NADH or NADP+/NADPH, and acofactor regenerating system comprising a ketoreductase and a lowersecondary alcohol, under suitable conditions where the compound offormula I is converted to the chiral amino acid compound of formula IIaand the lower secondary alcohol is converted to a ketone.

In the processes herein, the 2-oxo acid compound of formula I is asubstrate for the amino acid dehydrogenase. Accordingly, the R group inthe compound of formula I can be a substituted or unsubstituted:(C₁-C₁₀)alkyl, —(C₂-C₆)alkenyl, —(C₂-C₆)alkynyl, heteroalkyl,—(C₃-C₈)cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. Since aminoacid dehydrogenases are known to recognize naturally occurring aminoacids, the R group can be any side chain attached to the alpha carbon ofan amino acid of a naturally occurring amino acid. These include, amongothers, the following side chain structures shown in Table 1 (wheresquiggly line denotes point of connection of R group to rest ofmolecule):

TABLE 1

Other R groups recognized by amino acid dehydrogenases include, amongothers, the following structures shown in Table 2 (where squiggly linedenotes point of connection of R group to rest of molecule):

TABLE 2

In some embodiments, the amino acid dehydrogenase can be an L- orD-amino acid dehydrogenase. In other words, in the process forconversion of a pro-chiral 2-oxo acid of formula I to the amino acid offormula IIa, the amino acid dehydrogenase can be enantioselective forthe L- or D-amino acid. Thus, by selection of the appropriate amino aciddehydrogenase, the process of the present disclosure can be used toproduce the L- or D-amino acid in enantiomeric excess from the prochiral2-oxo acid. The amino acid dehydrogenase can be a naturally occurring,i.e., wild type, amino acid dehydrogenase or an engineered amino aciddehydrogenase. The engineered amino acid dehydrogenase can be selectedfor improved properties, such as increased enzymatic activity,thermostability, solvent stability, pH stability, co-factor preference,and/or altered substrate specificity.

In some embodiments of the process, the amino acid dehydrogenasecomprises a L-amino acid dehydrogenase and the chiral amino acidcompound of formula IIa is IIb,

having the indicated chirality. In this process, the chiral amino acidof formula IIb is formed in enantiomeric excess. In some embodiments,the chiral amino acid of formula IIb can be formed in at least 25%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% ormore in enantiomeric excess. In some embodiments, the chiral amino acidof formula IIb is formed in greater than 99% enantiomeric excess.

Various L-amino acid dehydrogenase can be obtained from organisms of thegenus Bacillus, Clostridium, Corynebacterium, Geobacillus,Natronobacterium, Synechocystis, Thermoactinomyces, Thermomicrobium,Carderia, Citrobacter, Proteus, and Pseudomonas, as well as frommammalian sources (e.g., beef liver). Specific organisms where theL-amino acid dehydrogenase can be obtained include, among others,Bacillus subtilis, Bacillus sphaericus, Bacillus stearothermophilus,Bacillus thermoproteoliticus, Brevibacterium sp., Clostridium symbiosum,Clostridium difficile, Geobacillus stearothermophilus, Natronobacteriummagadii, Synechocystis sp. PCC 6803, Thermoactinomyces intermedius,Citrobacter sp., Proteus sp., and Pseudomonas sp.

L-amino acid dehydrogenases useful in the process of the presentdisclosure capable of carrying out the conversion of an L-amino acid, inthe presence of an electron acceptor, to a 2-oxo acid, NH₃, and reducedacceptor. Suitable L-amino acid dehydrogenases have been described ine.g., Oshima et al., International Industrial Biotechnology 9 (1989)5-11; Oshima et al., European Journal of Biochemistry 191 (1990)715-720; Khan et al., Bioscience, Biotechnology and Biochemistry 69(2005) 1861-1870; Hummel et al., Applied Microbiology and Biotechnology26 (1987) 409-416 and Bommarius in Enzyme Catalysis in OrganicSynthesis, 2nd Edition (2002), ed. Drauz and Waldmann, Wiley-VCHWeinheim.

The polynucleotide and/or amino acid sequences of various L-amino aciddehydrogenases are known in the art and are available from known publicdatabases e.g., the GenBank (located at www.ncbi.nlm.nih.gov). In someembodiments, L-enantioselective amino acid dehydrogenases useful withthe process of the present disclosure have been described with respectto the type of amino acid acted upon/formed in the enzyme catalyzedprocess. Accordingly, in some embodiments, useful L-amino aciddehydrogenase references and sequence information can be obtained byentering into the indexed and searchable public databases any one of thefollowing enzyme classifications: L-alanine dehydrogenase (EC 1.4.1.1)(see e.g., Ohashima et al., 1979, Eur J Biochem. 100(1):29-30; Grimshawet al., 1981, Biochemistry. September 29; 20(20):5650-5), L-aspartatedehydrogenase (EC 1.4.1.21), L-erythro-3,5-diaminohexanoatedehydrogenase (EC 1.4.1.11), L-leucine dehydrogenase (EC 1.4.1.9),L-glutamate dehydrogenase (EC 1.4.1.2), glutamate dehydrogenase(NAD(P)+) (EC 1.4.1.3), glutamate dehydrogenase (NADP+) (EC 1.4.1.4),glycine dehydrogenase (EC 1.4.1.10), lysine dehydrogenase (EC 1.4.1.15),L-phenylalanine dehydrogenase (EC 1.4.1.20), L-serine dehydrogenase (EC1.4.1.7), L-valine dehydrogenase (EC 1.4.1.8), L-2,4-diaminopentanoatedehydrogenase, L-glutamate synthase, L-diaminopimelate dehydrogenase (EC1.4.1.12), L-N-methylalanine dehydrogenase, L-lysine 6-dehydrogenase,and L-tryptophan dehydrogenase, glutamate synthase (NADPH) (EC1.4.1.13); glutamate synthase (NADH>) (EC 1.4.1.14), diaminopimelatedehydrogenase (EC 1.4.1.16); N-methylalanine dehydrogenase (EC1.4.1.17), lysine 6-dehydrogenase (EC 1.4.1.18), and tryptophandehydrogenase (EC 1.4.1.19). The choice of the amino acid dehydrogenasecan be based on the type of oxo acid substrate recognized by the enzyme.

In some embodiments, the L-amino acid dehydrogenases are engineeredL-amino acid dehydrogenases in which mutations have been introduced intothe naturally occurring polypeptide to generate an enzyme with alteredproperties. Engineered L-amino acid dehydrogenases are described for, byway of example and not limitation, L-phenylalanine dehydrogenase (seee.g., Seah et al., 1995 FEBS Lett. 370(1-2):93-96; Busca et al., 2004,Org. Biomol. Chem. 2, 2684-2691.)

In some embodiments, the amino acid dehydrogenase comprises a D-aminoacid dehydrogenase, and the chiral amino acid of formula IIa is IIc,

having the indicated chirality. In this process, the chiral amino acidof formula IIc is formed in enantiomeric excess. In some embodiments,the chiral amino acid of formula IIc can be formed in at least 25%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% ormore in enantiomeric excess. In some embodiments, the chiral amino acidof formula IIc is formed in greater than 99% enantiomeric excess.

D-amino acid dehydrogenases can be obtained from Halobacterium,Methanosarcina, Pseudomonas, Pyrobaculum, Salmonella, Corynebacterium,and Escherichia. Specific species where the D-amino acid dehydrogenasecan be obtained include, among others, Pseudomonas aeruginosa,Pseudomonas fluorescens, Pyrobaculum islandicum, Salmonella typhimurium,Corynebacterium glutamicum, and Escherichia coli.

Like the L-amino acid dehydrogenases, references and sequences ofwild-type D-amino acid dehydrogenases are publicly available, forexample from the GenBank database available at the NCBI web-site. A fewexemplary wild-type D-amino acid dehydrogenase sequences listed byGenBank accession include: AAC36880 D-amino acid dehydrogenase[Escherichia coli] gi|145703|gb|AAC36880.1|[145703]; AAC36881 catabolicalanine racemase [Escherichia coli] gi| 145704|gb|AAC36881.1|[145704];AAC38139 D-amino acid dehydrogenase [Klebsiellaaerogenes]gi|2360965|gb|AAC38139.1|[2360965]; AAC74273 D-amino aciddehydrogenase [Escherichia coli str. K-12 substr. MG1655]gi|1787438|gb|AAC74273.1|[1787438]; AAD06449 D-Amino acid dehydrogenase[Helicobacter pylori J99] gi|4155445|gb|AAD06449.1|[4155445]; AAF40633D-amino acid dehydrogenase, small subunit [Neisseria meningitidis MC58]gi|7225395|gnl|tigr|NMB0176|gb|AAF40633.1|[7225395]; AAF83661 D-aminoacid dehydrogenase subunit [Xylella fastidiosa 9a5c]gi|9105759|gb|AAF83661.1|AE003925_1[9105759]; AAF93951 D-amino aciddehydrogenase, small subunit [Vibrio cholerae O1 biovar El Tor str.N16961]gi|9655235|gb|AAF93951.1∥gnl|TIGR|VC0786[9655235]; AAF951412,4-dienoyl-CoA reductase [Vibrio cholerae O1 biovar El Tor str. N16961]gi|9656535|gb|AAF95141.1∥gnl|TIGR|VC1993[9656535]; AAG08469 probableoxidoreductase [Pseudomonas aeruginosa PAO1]gi|9951378|gb|AAG08469.1|AE004921_5|gnl|PseudoCAP|PA5084[9951378];AAG08689 D-amino acid dehydrogenase, small subunit [Pseudomonasaeruginosa PAO1]gi|9951620|gb|AAG08689.1|AE004943_5|gnl|PseudoCAP|PA5304[9951620];AAG56040 D-amino acid dehydrogenase subunit [Escherichia coli O157:H7EDL933] gi∥2514889|gb|AAG56040.1|AE005336_1 [12514889]; AAK90026 D-aminoacid dehydrogenase [Agrobacterium tumefaciens str. C58] gi|15159999|gb|AAK90026.1|[15159999]; AAK90097 D-amino acid dehydrogenase,small subunit [Agrobacterium tumefaciens str. C58]gi|15160086|gb|AAK90097.1|[15160086]; AAL20718 D-amino aciddehydrogenase subunit [Salmonella enterica subsp. enterica serovarTyphimurium str. LT2] gi| 6420334|gb|AAL20718.1|[16420334]; AAL53615d-amino acid dehydrogenase small subunit [Brucella melitensis bv. 1 str.16M] gi|17984529|gb|AAL53615.1∥gnl|integgen|BMEII0373[17984529];AAL73201 putative D-amino acid dehydrogenase [Agrobacterium sp. IPI-671] gi| 8478564|gb|AAL73201.11AF335479_5[18478564]; AAM38531 D-aminoacid dehydrogenase subunit [Xanthomonas axonopodis pv. citri str. 306]gi|21110075|gb|AAM38531.1∥gnl|unicamp|XAC3688 [21110075]; AAM42918D-amino acid dehydrogenase subunit [Xanthomonas campestris pv.campestris str. ATCC 33913]gi|21114929|gb|AAM42918.1∥gnl|unicamp|XCC3648[21114929]; AAM85736D-amino acid dehydrogenase subunit [Yersinia pestis KIM 10]gi|21959016|gb|AAM85736.1|AE013821_2[21959016]; AAN34096 D-alaninedehydrogenase, small subunit [Brucella suis 1330]gi|23464296|gnl|tigr|BRA0924|gb|AAN34096.11 [23464296]; AAN42793 D-aminoacid dehydrogenase subunit [Shigella flexneri 2a str. 301]gi|56383395|gb|AAN42793.2∥gnl|mgcchina|SF0001178[56383395]; and AAN69891D-amino acid dehydrogenase, small subunit, putative [Pseudomonas putidaKT2440] gi|124986030|gb|AAN69891.1|AE016628_4|gnl|tigr|PP4311[24986030].

In some embodiments, the D-amino acid dehydrogenase is selected fromD-alanine dehydrogenase (e.g., gi|23464296|gb|AAN34096.1| D-alaninedehydrogenase, small subunit [Brucella suis 1330]), D-threoninedehydrogenase (e.g., gi|3845577|dbj|BAA34184.1| D-threoninedehydrogenase [Pseudomonas cruciviae]), D-proline dehydrogenase (e.g.,gi|145283977|gb|ABP51559.1| D-proline dehydrogenase [Pyrobaculumarsenaticum DSM 13514]).

In some embodiments, the D-amino acid dehydrogenases are engineeredD-amino acid dehydrogenases in which mutations have been introduced intothe naturally occurring polypeptide to generate an enzyme with alteredenzyme properties. Engineered D-amino acid dehydrogenases are describedin e.g., Vedha-Peters et al., “Creation of a Broad-Range and HighlyStereoselective D-Amino Acid Dehydrogenase for the One-Step Synthesis ofD-Amino Acids” J. Am. Chem. Soc. 2006, 128, 10923-10929, or in U.S. Pat.No. 7,550,277, which is hereby incorporated by reference herein.

In the process mediated by the amino acid dehydrogenases, the reactionmedium contains an ammonium source, which provides the NH₃ group forformation of the amino acid of formula IIa from the 2-oxo acid offormula I. Any compound which is suitable for this purpose can be usedas the ammonium source. Exemplary compounds include, among others,ammonium salts, such as ammonium halide (e.g., ammonium chloride),ammonium formate, ammonium sulfate, ammonium phosphate, ammoniumnitrate, ammonium tartrate, and ammonium acetate.

While the process described herein is generally used for the preparationof L- or D-amino acids, the amino acid dehydrogenase can also carry outthe reverse reaction, i.e., conversion of an amino acid to itscorresponding 2-oxo acid. When the substrate is an enantiomerically pureL- or D-amino acid, an appropriate amino acid dehydrogenase can be usedfor the conversion of the amino acid to the 2-oxo acid. For instance, anL-amino acid dehydrogenase is selected for conversion of L-amino acidpreparations to the corresponding 2-oxo acid.

In some embodiments, the stereospecificity of amino acid dehydrogenasescan be exploited for the chiral resolution of mixtures of L- and D-aminoacids. For example, an L-amino acid dehydrogenase can be used tostereospecifically convert the L-amino acid in a mixture of L- andD-amino acids to the corresponding 2-oxo acid, thereby resulting in acomposition that has the D-amino acid in enantiomeric excess. Similarly,a D-amino acid dehydrogenase can be used to stereospecifically convertthe D-amino acid in a mixture of L- and D-amino acids to thecorresponding 2-oxo acid, thereby resulting in a composition that hasthe L-amino acid in enantiomeric excess. When desired, the amino acidpresent in enantiomeric excess can be isolated from the product mixture.

Accordingly, in some embodiments, the present disclosure provides aprocess for converting a compound mixture of formula IId, whichcomprises a substrate for an amino acid dehydrogenase, to a compositionof a 2 oxo acid compound of formula I and a chiral amino acid of formulaIIa:

having a chiral carbon marked with an *, where the compound mixture offormula IId comprises L- and D-amino acid compounds of formula IIb andIIc and the chiral amino acid of formula IIa is an L- or D-amino acid.In these embodiments, the process for chiral resolution of a mixture ofL- and D-amino acids can comprise contacting the compound mixture offormula IId with a reaction medium comprising an enantioselective aminoacid dehydrogenase, NAD+/NADH or NADP+/NADPH, and a cofactor recyclingsystem comprising a ketoreductase and a lower alkyl ketone, underconditions where either the L-amino acid or D-amino acid of the compoundmixture of formula IId is converted to a compound of formula I therebyresulting in an enantiomeric excess of the amino acid of formula IIa(which the chiral amino acid not converted by the amino aciddehydrogenase), and the lower alkyl ketone is converted to a lowersecondary alcohol.

In some embodiments of the process, the compound mixture of IId is aracemic mixture of L- and D-amino acid compounds of formulas IIb andIIc, as represented by formula IIe:

In some embodiments, the process can be used for the chiral resolutionof a mixture of L- and D-amino acids to form a chiral amino acid offormula IIc:

having the indicated chirality in enantiomeric excess. In theseembodiments, the process can comprise contacting the compound mixture offormula IId (which can include a racemic mixture of formula IIe) with areaction medium comprising an L-amino acid dehydrogenase, NAD+/NADH orNADP+/NADPH and a cofactor recycling system comprising a ketoreductaseand a lower alkyl ketone, under conditions where the chiral amino acidcompound of formula IIb in the compound mixture of formula IId isconverted to the 2-oxo acid compound of formula I thereby resulting inan enantiomeric excess of the chiral amino acid of formula IIc, and thelower alkyl ketone is converted to the corresponding lower secondaryalcohol. In some embodiments, the chiral amino acid of formula IIc canbe formed in at least 25%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% or more in enantiomeric excess. In someembodiments, the chiral amino acid of formula IIc is formed in greaterthan 99% enantiomeric excess.

In the process for chiral resolution of a mixture of L- and D-aminoacids to form the D-amino acid in enantiomeric excess, the L-amino aciddehydrogenase can be from Bacillus, Clostridium, Corynebacterium,Geobacillus, Natronobacterium, Synechocystis, Thermoactinomyces,Thermos, Thermomicrobium, or Carderia.

In some embodiments, the L-amino acid dehydrogenase can be selected fromL-alanine dehydrogenase, L-aspartate dehydrogenase,L-erythro-3,5-diaminohexanoate dehydrogenase, L-leucine dehydrogenase,L-glutamate dehydrogenase, lysine dehydrogenase, L-phenylalaninedehydrogenase, L-serine dehydrogenase. L-valine dehydrogenase,L-2,4-diaminopentanoate dehydrogenase, L-glutamate synthase,L-diaminopimelate dehydrogenase, L-N-methylalanine dehydrogenase,L-lysine 6-dehydrogenase, and L-tryptophan dehydrogenase, as describedabove.

In some embodiments, the process can be used for the chiral resolutionof a mixture of L- and D-amino acids to form the chiral amino acid offormula IIb,

having the indicated chirality in enantiomeric excess. In theseembodiments, the process can comprise contacting the compound mixture offormula IId (which can include a racemic mixture of formula IIe) with areaction medium comprising a D-amino acid dehydrogenase, NAD+/NADH orNADP+/NADPH and a cofactor recycling system comprising a ketoreductaseand a lower alkyl ketone, under conditions where the chiral amino acidof formula IIc in the compound mixture of formula IId is converted tothe 2-oxo acid compound of formula I thereby resulting in anenantiomeric excess of the chiral amino acid of formula IIb, and thelower alkyl ketone is converted to the corresponding lower secondaryalcohol. In some embodiments, the chiral amino acid of formula IIb canbe formed in at least 25%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% or more in enantiomeric excess. In someembodiments, the chiral amino acid of formula IIb is formed in greaterthan 99% enantiomeric excess.

In the process for chiral resolution of a mixture of L- and D-aminoacids to form the L-amino acid in enantiomeric excess, the D-amino aciddehydrogenase can be from Halobacterium, Methanosarcina, Pseudomonas,Pyrobaculum, Salmonella, Corynebacterium, or Escherichia.

In some embodiments of the process herein, the amino acid dehydrogenasecan be present in the form of whole cells, including whole cellstransformed with polynucleotide constructs expressing wild type orengineered amino acid dehydrogenases. In some embodiments, the aminoacid dehydrogenase can be present in the form of cell extracts and/orlysates thereof, and may be employed in a variety of different forms,including solid (e.g., lyophilized, spray-dried, and the like) orsemisolid (e.g., a crude paste). In some embodiments, the amino aciddehydrogenase is isolated, and can be in a substantially purified form.In some embodiments of the process, both the amino acid dehydrogenaseand the ketoreductase of the regenerating system can be present in theform of whole cells, including whole cells transformed withpolynucleotide constructs such that the whole cells express both theamino acid dehydrogenase and the ketoreductase.

In the embodiments of the processes disclosure herein, the amino aciddehydrogenase is used in combination with cofactor regenerating systemcomprising: a ketoreductase capable of reducing NAD⁺ and/or NADP⁺ toNADH and NADPH, respectively, and an alcohol (e.g., a lower secondaryalcohol) that is a substrate for the ketoreductase. In the reduction ofthe cofactor, the ketoreductase converts the alcohol to thecorresponding carbonyl compound (e.g., a lower alkyl ketone).

In the embodiments herein, the ketoreductase can be a wild typeketoreductase, or an engineered ketoreductase, in particular anengineered ketoreductase with an improved enzyme property. As usedherein, a ketoreductase enzyme that has an “improved enzyme property”refers to a ketoreductase enzyme that exhibits an improvement in anyenzyme property as compared to a reference ketoreductase enzyme. For theengineered ketoreductase enzymes described herein, the comparison isgenerally made to the wild-type ketoreductase enzyme, although in someembodiments, the reference ketoreductase can be an improved engineeredketoreductase. In some embodiments, the ketoreductase is an engineeredketoreductase characterized by increased thermostability, increasedsolvent stability, increased pH stability, and/or increased enzymaticactivity relative to the wild type ketoreductase.

In some embodiments of the process, the ketoreductase used in thecofactor recycling system has an improved property over a referenceketoreductase of increased activity in the conversion of the lowersecondary alcohol (e.g., isopropanol) of the recycling system to thecorresponding lower alkyl ketone. In some embodiments, the ketoreductasehaving the increased activity in the conversion of the lower secondaryalcohol is at least 2.0 fold, 2.5 fold, 5.0 fold, 7.5 fold, 10-fold, ormore improved relative to a reference ketoreductase. In someembodiments, the ketoreductase is an engineered ketoreductase derivedfrom the wild-type ketoreductase of Novosphingobium aromaticivorans(e.g., gi|1145322460|gblABP64403.11 [145322460]).

The improved activity of engineered ketoreductases (derived fromNovosphingobium aromaticivorans ketoreductase of SEQ ID NO: 2) for theconversion of the secondary alcohol, isopropanol (IPA) to itscorresponding product, acetone was determined relative to the sameactivity for the reference ketoreductase of SEQ ID NO: 2. Relative IPAactivity was determined using an assay with the following reactionconditions: 100 μl 10× diluted engineered KRED lysate, 10% IPA (v/v),0.5 g/L NAD⁺, 100 mM TEA, pH 7.5. Exemplary engineered ketoreductasesexhibiting at least 2-fold increased activity with IPA relative to SEQID NO: 2 are listed in Table 3. The fold-improvement in IPA activityrelative to SEQ ID NO: 2 was quantified as follows: “+” indicates atleast 200% to 250% improvement; “++” indicates <250% to 500%improvement; and “+++” indicates >500% to 1000% improvement; and “++++”indicates >1000% to 2000% improvement.

TABLE 3 SEQ ID FIOP in IPA NO: activity 2 [control] 4 + 6 ++ 8 ++ 10 ++12 +++ 14 ++ 16 + 18 ++++ 20 ++ 24 +++

In some embodiments, the ketoreductase having the increased activity inthe conversion of the lower secondary alcohol is an engineeredketoreductase comprising an amino acid sequence having at least 80%,85%, 90%, 95%, 98%, 99%, or more identity to a sequence selected fromSEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24. In someembodiments, the ketoreductase having the increased activity in theconversion of the lower secondary alcohol is an engineered ketoreductasecomprising an amino acid sequence selected from SEQ ID NO: 4, 6, 8, 10,12, 14, 16, 18, 20, 22, and 24.

In some embodiments of the process, the ketoreductase used in thecofactor recycling system has an improved property over a referenceketoreductase of decreased or no activity with the 2-oxo acid compoundof formula I (e.g., trimethylpyruvic acid) which is a substrate for theamino acid dehydrogenase used in the process. In some embodiments of theprocess, the activity of the ketoreductase used in the cofactorrecycling system with the compound of formula I is less than about 5%,2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or an even smaller percentage of theactivity of the amino acid dehydrogenase used in the process with thecompound of formula I. In some embodiments of the process, theketoreductase used in the cofactor recycling system has no detectableactivity with the 2-oxo acid compound of formula I.

The relative IPA activity of each of the engineered ketoreductases shownin Table 3 was measured under the same conditions in the presence of thecompound of formula I, trimethylpyruvic acid (10% IPA (v/v), 0.5 g/LNAD+, 1 mM TMP, 100 mM TEA pH 7.5, 100 μl 10× KRED diluted lysate). Lessthan 5% increase or decrease was seen in the presence of the TMP,indicating that the engineered ketoreductases listed in Table 3 did notuse TMP as a substrate.

Various ketoreductases that can be used in the cofactor regeneratingsystem include ketoreductases from, by way of example and notlimitation, bacteria, such as the genus Escherichia, the genus Bacillus,the genus Pseudomonas, the genus Serratia, the genus Brevibacterium, thegenus Corynebacterium, the genus Streptococcus, the genus Lactobacillus,the genus Novosphingobium; actinomycetes such as the genus Rhodococcus,the genus Streptomyces; yeasts such as the genus Saccharomyces, thegenus Kluyveromyces, the genus Thermoanerobium, the genusSchizosaccharomyces, the genus Sporobolomyces, the genusZygosaccharomyces, the genus Yarrowia, the genus Trichosporon, the genusRhodosporidium, the genus Pichia, the genus Candida; and fungi such asthe genus Neurospora, the genus Aspergillus, the genus Cephalosporium,the genus Trichoderma.

In some embodiments, the ketoreductase can be derived from Lactobacilluskefir, Lactobacillus brevis, Lactobacillus minor, Candida sonorensis,Candida boidini, Candida guilliermondi, Candida magnoliae, Candidautilis, Candida maltosa, Candida kefir, Candida parapslosis, Geotrichumcandidum, Rhodococcus erythropolis, Rhodotorula glutinis, Hansenulafabianii, Hansenula polymorpha, Hansenula saturnus, Nocardiasalmonicolor, Novosphingobium aromaticivorans, Pichia anomala, Pichiacapsulata, Pichia membranafaciens, Pichia methanolica, Pichia pinus,Pichia silvicola, Pichia stipitis, Sphingomonas paucimobilis,Sporobolomyces salmonicolor, Streptomyces coelicolor, Thermoanerobiumbrockii, or Saccharomyces cerevisiae.

In some embodiments, the ketoreductase is a wild-type ketoreductaselisted in Table 4 or an engineered ketoreductase derived from awild-type ketoreductase listed in Table 4.

TABLE 4 Wild type KRED from various microorganisms Microorganism GenbankAcc. No. GI No. Reference Candida magnoliae AB036927.1 12657576 SEQ IDNo 2 in US publ. no. 20060195947A1 Saccharomyces cerevisiae NP_010159.16320079 SEQ ID NO: 110 in US publ. no. 20090191605A1 Lactobacillusbrevis 1NXQ_A 30749782 SEQ ID NO: 2 in US publ. no. 20090191605A1Rhodococcus erythropolis AAN73270.1 34776951 SEQ ID NO: 112 in US publ.no. 20090191605A1 Saccharomyces cerevisiae NP_011476 6321399 SEQ ID NO:114 in US publ. no. 20090191605A1 Saccharomyces cerevisiae NP_010656.16320576 SEQ ID NO: 116 in US publ. no. 20090191605A1 Saccharomycescerevisiae NP_014490.1 6324421 SEQ ID NO: 118 in US publ. no.20090191605A1 Lactobacillus kefir AAP94029.1 33112056 SEQ ID NO: 4 in USpubl. no. 20090191605A1 Sporobolomyces salmonicolor Q9UUN9 30315955 SEQID No 104 in US publ. no. 20090191605A1 Streptomyces coelicolorNP_631415.1 21225636 SEQ ID No 102 in US publ. no. 20090191605A1Thermoanaerobium brockii X64841.1 1771790 SEQ ID No 108 in US publ. no.20090191605A1 Candida parapsilosis BAA24528 2815409 Julich ChiralSolutions Cat. No. 03.11 Lactobacillus brevis ABJ63353.1 116098204Julich Chiral Solutions Cat. No. 8.1 Candida boidinii CAD66648 28400789Julich Chiral Solutions Cat. No. 02.10 Lactobacillus leichmannii FlukaCat. No. 61306

In some embodiments, the ketoreductase is from Lactobacillus, such as,among others, Lactobacillus kefir, Lactobacillus brevis, orLactobacillus minor. Wild type ketoreductase from Lactobacillus kefir isdescribed in Genbank accession no. AAP94029 GI:33112056. Wild typeketoreductase from Lactobacillus brevis is described in CAD66648GI:28400789.

In some embodiments, the ketoreductase is an engineered ketoreductasederived from a wild type ketoreductase of Lactobacillus. Engineeredketoreductases of Lactobacillus, for example, L. kefir, L. brevis, andL. minor, with improved enzyme properties are described in US patentpublications US20080318295, US20090093031, US20090191605, US20090155863,US20090162909, U.S. Ser. No. 12/545,034, filed Aug. 20, 2009, U.S. Ser.No. 12/545,761, filed Aug. 21, 2009, U.S. Ser. No. 12/549,154, filedAug. 27, 2009, and U.S. Ser. No. 12/549,293, filed Aug. 27, 2009, ofwhich each of the ketoreductase polypeptides disclosed therein arehereby incorporated by reference herein.

In some embodiments, the ketoreductase of Candida is from Candidamagnoliae. Wild type ketoreductase of Candida magnoliae is described ine.g., Wada et al., Biosci. Biotechnol. Biochem. 62(2): 280-285 (1998).Engineered ketoreductases with improved enzyme properties derived fromCandida magnoliae ketoreductase is described in US20060195947, of whicheach of the ketoreductase polypeptides disclosed therein is herebyincorporated by reference herein.

In some embodiments, the ketoreductase of Saccharomyces is fromSaccharomyces cerevisiae. Wild type ketoreductase from Saccharomycescerevisiae is described in US20080248539. Engineered ketoreductases withimproved enzyme properties derived from Saccharomyces cerevisiaeketoreductase are described in US20080248539. Each of the ketoreductasepolypeptides disclosed therein are incorporated by reference herein.

In some embodiments, the ketoreductase of Novosphingobium is fromNovosphingobium aromaticivorans. A wild type ketoreductase gene fromNovosphingobium aromaticivorans is provided as GenBank accessionCP000677.1, and the encoded polypeptide sequence is accession no.gi|145322460|gb|ABP64403.1|[145322460]. Engineered ketoreductasesderived from Novosphingobium aromaticivorans wild type are described inU.S. provisional application 61/219,162, filed Jun. 22, 2009, which ishereby incorporated by reference herein. Exemplary engineeredpolynucleotides and the corresponding ketoreductase polypeptides derivedfrom the Novosphingobium aromaticivorans wild type and having theimproved property of increased activity in converting isopropanol toacetone are presented in the sequence listing incorporated herein as SEQID NO: 3-24. In some embodiments, the engineered ketoreductase comprisesan amino acid sequence selected from the group consisting of SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24.

In some embodiments, the ketoreductase used in the process is capable ofrecycling cofactor by converting isopropanol (IPA) to acetone in areaction medium of 3 to 20% IPA at a pH of about 9.0 to 10.5 with anactivity at least 1.5-fold greater than the reference ketoreductase ofSEQ ID NO: 2.

In some embodiments, the ketoreductase can be present in the form ofwhole cells, including whole cells transformed with polynucleotideconstructs expressing wild type or engineered ketoreductases. In someembodiments, the ketoreductase can present as cell extracts and/orlysates thereof, and may be employed in a variety of different forms,including solid (e.g., lyophilized, spray-dried, and the like) orsemisolid (e.g., a crude paste). In some embodiments of the process, theketoreductase is isolated, and can be in substantially purified form.

In the cofactor regenerating process carried out by the ketoreductase,an alcohol is used as the substrate reductant for the generation ofreduced cofactor NADH or NADPH. As noted above, while primary alcoholssubstrates recognized by the ketoreductase, such as ethanol, can beused, preferable are secondary alcohols, particularly lower secondaryalcohols. Suitable secondary alcohols include lower secondary alkanolsand aryl-alkyl carbinols. Examples of lower secondary alcohols includeisopropanol, 2-butanol, 3-methyl-2-butanol, 2-pentanol, 3-pentanol,3,3-dimethyl-2-butanol, and the like. Suitable aryl-alkyl carbinolsinclude unsubstituted and substituted 1-arylethanols. In someembodiments, the secondary alcohol is isopropanol.

The alcohol, particularly a secondary alcohol (e.g., isopropanol), canbe present at about 1% to 60% v/v, about 1% to 50% v/v, about 1% to 40%v/v, about 1% to 30% v/v, about 1% to 20% v/v, or about 1% to 10% v/v ofthe reaction medium. In some embodiments, the alcohol can be present atabout 10% to 60% v/v, about 10% to 50% v/v, about 10% to 40% v/v, about10% to 30% v/v, or about 10% to 20% v/v of the reaction medium. In someembodiments, the alcohol can be present at about 20% to 60% v/v, about20% to 50% v/v, about 20% to 40% v/v, or about 20% to 30% v/v of thereaction medium. The amount of alcohol useful in the process can bedetermined based on the activities of the amino acid dehydrogenase andthe ketoreductase in the presence of a defined amount of alcohol.

In some embodiments of the process, the product formed from theketoreductase can be removed from the reaction medium to improveconversion of the alcohol to the corresponding carbonyl product, andthereby pushing the equilibrium of the process to the reduction of NAD+or NADP+ to NADH or NADPH. For instance, where the carbonyl product isvolatile, the product can be removed by sparging the reaction mediumwith a non-reactive gas or by lowering the vapor pressure of thereaction medium and removing the volatile carbonyl product. In someembodiments of the process, the alcohol substrate for the ketoreductaseis a lower secondary alcohol, and the corresponding lower alkyl ketoneformed from the lower secondary alcohol is removed from the reactionmedium. In some embodiments, where the alcohol is isopropanol, theproduct acetone can be removed by sparging the reaction medium with anon-reactive gas, such as nitrogen, or by applying a vacuum to thereaction medium and removing the acetone by condensation.

Where appropriate for use in the processes, the amino aciddehydrogenases and ketoreductase enzymes present in cells, such asengineered enzymes expressed in host cells, can be recovered from thecells and or the culture medium using any one or more of the well knowntechniques for protein purification, including, among others, lysozymetreatment, sonication, filtration, salting-out, ultra-centrifugation,and chromatography. Suitable solutions for lysing and the highefficiency extraction of proteins from bacteria, such as E. coli, arecommercially available under the trade name CelLytic B™ fromSigma-Aldrich of St. Louis Mo. The cell extracts or cell lysates may bepartially purified by precipitation (ammonium sulfate,polyethyleneimine, heat treatment or the like, followed by a desaltingprocedure prior to lyophilization (e.g., ultrafiltration, dialysis, andthe like). Any of the cell preparations may be stabilized bycrosslinking using known crosslinking agents, such as, for example,glutaraldehyde or immobilization to a solid phase (e.g., Eupergit C, andthe like).

The reactions described herein are generally carried out in a solvent.Suitable solvents include water, organic solvents (e.g., ethyl acetate,butyl acetate, 1-octanol, heptane, octane, methyl t-butyl ether (MTBE),toluene, and the like), and ionic liquids (e.g., 1-ethyl4-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazoliumtetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, andthe like). In some embodiments, aqueous solvents, including water andaqueous co-solvent systems, can be used.

Exemplary aqueous co-solvent systems have water and one or more organicsolvent. In general, an organic solvent component of an aqueousco-solvent system is selected such that it does not significantlyinactivate the enzymes (i.e., amino acid dehydrogenase andketoreductase). Appropriate co-solvent systems can be readily identifiedby measuring the enzymatic activity of the specified enzyme with adefined substrate of interest in the candidate solvent system, utilizingan enzyme activity assay, such as those described herein.

The organic solvent component of an aqueous co-solvent system may bemiscible with the aqueous component, providing a single liquid phase, ormay be partly miscible or immiscible with the aqueous component,providing two liquid phases. Generally, when an aqueous co-solventsystem is employed, it is selected to be biphasic, with water dispersedin an organic solvent, or vice-versa. Generally, when an aqueousco-solvent system is utilized, it is desirable to select an organicsolvent that can be readily separated from the aqueous phase. Ingeneral, the ratio of water to organic solvent in the co-solvent systemis typically in the range of from about 90:10 to about 10:90 (v/v)organic solvent to water, and between 80:20 and 20:80 (v/v) organicsolvent to water. The co-solvent system may be pre-formed prior toaddition to the reaction mixture, or it may be formed in situ in thereaction vessel.

During the course of the amino acid dehydrogenase and ketoreductasemediated process, the pH of the reaction medium may change. The pH ofthe reaction medium may be maintained at a desired pH or within adesired pH range by the addition of an acid or a base during the courseof the reaction. Alternatively, the pH may be controlled by using anaqueous solvent that comprises a buffer. Suitable buffers to maintaindesired pH ranges are known in the art and include, for example,phosphate buffer, triethanolamine buffer, and the like. Combinations ofbuffering and acid or base addition may also be used. Typically, basesadded to unbuffered or partially buffered reaction mixtures over thecourse of the reduction are added in aqueous solutions.

In carrying out embodiments of the process described herein, either theoxidized or reduced form of the cofactor may be provided initially. Asdescribed above, the cofactor regenerating system converts oxidizedcofactor to its reduced form (or vice-versa), which is then utilized inthe reduction (or oxidation) of the substrate.

The solid reactants (e.g., enzyme, salts, etc.) may be provided to thereaction in a variety of different forms, including powder (e.g.,lyophilized, spray dried, and the like), solution, emulsion, suspension,and the like. The reactants can be readily lyophilized or spray driedusing methods and equipment that are known to those having ordinaryskill in the art. For example, the protein solution can be frozen at−80° C. in small aliquots, then added to a pre-chilled lyophilizationchamber, followed by the application of a vacuum. After the removal ofwater from the samples, the temperature is typically raised to about 4°C. before release of the vacuum and retrieval of the lyophilizedsamples.

The quantities of reactants used in the reduction reaction willgenerally vary depending on the quantities of product desired, andconcomitantly, the amount of substrate employed. The followingguidelines can be used to determine the amounts of amino aciddehydrogenase, ketoreductase, and cofactor. Those having ordinary skillin the art will readily understand how to vary these quantities totailor them to the desired level of productivity and scale ofproduction. Appropriate quantities of cofactor regenerating system maybe readily determined by routine experimentation based on the amount ofamino acid dehydrogenase and ketoreductase utilized.

The order of addition of reactants is not critical. The reactants may beadded together at the same time to a solvent (e.g., monophasic solvent,biphasic aqueous co-solvent system, and the like), or alternatively,some of the reactants may be added separately, and some together atdifferent time points.

For improved mixing efficiency when an aqueous co-solvent system isused, the amino acid dehydrogenase and the ketoreductase may be addedand mixed into the aqueous phase first. The organic phase may then beadded and mixed in, followed by addition of the enzyme substrates.Alternatively, the enzymes may be premixed in the organic phase, priorto addition to the aqueous phase.

Suitable conditions for carrying out the amino aciddehydrogenase/ketoreductase mediated process described herein include awide variety of conditions which can be optimized by routineexperimentation that includes, but is not limited to, contacting theenzymes and substrates at an experimental pH and temperature anddetecting product, for example, using the methods described in theExamples provided herein.

Generally, the process can be carried out at a pH of about 11 or below,usually in the range of from about 8.0 to about 11. In some embodiments,the process may be carried out a neutral pH, i.e., about 7.0. Theoptimal pH of the reaction medium can be determined based on the pHsensitivities of the amino acid dehydrogenase and ketoreductase enzymes.In some embodiments, the process is carried out at a pH of about 9.5 orbelow, usually in the range of from about 8.5 to about 9.5, and in someembodiments at a pH of about 8.75 to about 9.25, and in some embodimentsthe process is carried out at about pH 9. In some embodiments, theprocess may can be carried out at a pH of about 9 to about 11,particularly at about pH 10 to about 11.

In some embodiments, the process described herein can be carried out ata temperature in the range of from about 15° C. to about 75° C. In someembodiments, the reaction is carried out at a temperature in the rangeof from about 20° C. to about 55° C. In some embodiments, it is carriedout at a temperature in the range of from about 20° C. to about 45° C.In some embodiments, the process is carried out at a temperature ofabout 35° C. to about 40° C. The reaction may also be carried out underambient conditions.

The process is generally allowed to proceed until there is no furtherconversion or substantial conversion of substrate (e.g., oxo acid) tothe product (e.g., amino acid) or until there is essentially complete,or nearly complete, conversion of substrate to product. Conversion ofsubstrate to product can be monitored using known methods by detectingsubstrate and/or product. Suitable methods include gas chromatography,HPLC, and the like. Conversion yields of the product amino acidgenerated in the reaction mixture can be greater than about 50%, greaterthan about 60%, greater than about 70%, greater than about 80%, greaterthan 90%, and are often greater than about 97%.

In some embodiments, the process described herein can be used in theconversion of a compound of formula I which is3,3-dimethyl-2-oxobutanoic acid (also referred to herein as“trimethylpyruvic acid” or “TMP”) to the chiral amino acid(S)-2-amino-3,3-dimethylbutanoic acid (also referred to herein as“L-tert-leucine”), where the amino acid dehydrogenase comprises aL-leucine dehydrogenase (“LeuDH”), as illustrated by Scheme 2.

The chiral amino acid L-tert-leucine is useful in the synthesis ofintermediates, particularly advanced pharmaceutical intermediate used inthe preparation of drug compounds, e.g., atazanavir, boceprevir, andtelaprevir.

In some embodiments, the process for producing L-tert-leucine cancomprise contacting the substrate 3,3-dimethyl-2-oxobutanoic acid with areaction medium comprising a L-leucine dehydrogenase, an ammonium iondonor, NAD+/NADH or NADP+/NADPH, and a cofactor regenerating systemcomprising a ketoreductase and an alcohol, under suitable reactionconditions to convert the 3,3-dimethyl-2-oxobutanoic acid to product(S)-2-amino-3,3-dimethylbutanoic acid, and the alcohol to thecorresponding carbonyl compound. In particular, the alcohol is asecondary alcohol, as described herein.

In some embodiments, the L-leucine dehydrogenase used in the process canbe a wild type leucine dehydrogenase or an engineered leucinedehydrogenase. L-leucine dehydrogenases can be from genus Bacillus,Clostridium, Corynebacterium, Geobacillus, Natronobacterium,Thermoactinomyces, Thermos, Thermomicrobium, or Carderia.

In some embodiments, the leucine dehydrogenase is from Bacillusacidokaludarius, Bacillus brevis, Bacillus caldolyticus, Bacilluscereus, Bacillus megaterium, Bacillus mesentericus, Bacillus mycoides,Bacillus natto, Bacillus pumilus, Bacillus sphaericus, Bacillusstearothermophilus, Bacillus subtilis, Clostridium thermoaceticum,Corynebacterium pseudodiphtheriticum, Geobacillus stearothermophilus,Natronobacterium magadii, or Thermoactinomyces intermedius.

The process disclosed herein could be used with any wild-type L-leucinedehydrogenase. Sequences of such wild-type enzymes are publiclyavailable, for example from the GenBank database available at the NCBIweb-site.

A few exemplary wild-type leucine dehydrogenase enzyme sequences listedby GenBank accession include: leucine dehydrogenase [Geobacillusstearothermophilus] gi|34014423|dbj|BAC81829.1|; leucine dehydrogenase[Geobacillus stearothermophilus] gi|143145|gb|M22977.1|; leucinedehydrogenase [Geobacillus stearothermophilus]gi|34014421|dbj|AB103384.1|; leucine dehydrogenase [Bacilluslicheniformis] gi|1477946|gb|AAB36205.1∥bbm|385403|bbs|177171 [1477946];LEUCINE-DEHYDROGENASE [Bacillus cereus]gi|6741939|emb|CAB69610.1|[6741939]; leucine dehydrogenase[Streptosporangium roseum DSM 43021]gi|2719701731gnl|REF_jgi|Sros_8995|ref|YP_003344369.1|[271970173];leucine dehydrogenase [Streptosporangium roseum DSM 43021]gi|270513348|gnl|jgi|Sros_8995|gb|ACZ91626.1|[270513348]; leucinedehydrogenase [Natranaerobius thermophilus JW/NM-WN-LF]gi|179351985|gnl|jgi|Nther_2701|gb|ACB86255.1|[179351985]; leucinedehydrogenase [Natranaerobius thermophilus JW/NM-WN-LF]gi|179351644|gnl|jgi|Nther_2349|gb|ACB85914.1|[179351644]; leucinedehydrogenase [Natranaerobius thermophilus JW/NM-WN-LF] gi|179350985|gnl|jgi|Nther_1681|gb|ACB85255.1|[179350985]; leucinedehydrogenase [Shewanella amazonensis SB2B]gi|119767702|gnl|jgi|Sama_2067|gb|ABM00273.1|[119767702]; leucinedehydrogenase [Shewanella sp. ANA-3]gi|117612521|gnl|jgi|Shewana3_1742|gb|ABK47975.1|[117612521]; leucinedehydrogenase [Shewanella frigidimarina NCIMB 400] gi|114334729|gnl|jgi|Sfri_2265|gb|ABI72111.1|[114334729]; leucinedehydrogenase [Shewanella sp. MR-7]gi|113888616|gnl|jgi|Shewmr7_1673|gb|ABI42667.1|[113888616]; leucinedehydrogenase [Shewanella sp. MR-4]gi|113884623|gnl|jgi|Shewmr4_1598|gb|ABI38675.1|[113884623]; leucinedehydrogenase [Chelativorans sp. BNC1]gi|110286554|gnl|jgi|Meso_3241|gb|ABG64613.1|[110286554]; leucinedehydrogenase [Pseudoalteromonas atlantica T6c]gi|109701583|gnl|jgi|Patl_2995|gb|ABG41503.1|[109701583]; leucinedehydrogenase [Rubrobacter xylanophilus DSM 9941]gi|108766512|gnl|jgi|Rxyl_2467|gb|ABG05394.1|[108766512]; leucinedehydrogenase [Chlamydophila pneumoniae TW-183]gi|33236793|gb|AAP98880.1∥gnl|byk|CpB0951 [33236793]; Leucinedehydrogenase [Bacillus cereus m1293]gi1229198293|ref|ZP_04325000.1∥gnl|WGS:NZ_ACLS01|bcere0001_38230[229198293];Leucine dehydrogenase [Bacillus cereus ATCC 10876]gi|229192377|ref|ZP_04319341.1∥gnl|WGS:NZ_ACLT01|bcere0002_40300[229192377];Leucine dehydrogenase [Bacillus cereus BGSC 6E1]gi|229186408|ref|ZP_04313572.1∥gnl|WGS:NZ_ACLU01|bcere0004_39530[229186408];Leucine dehydrogenase [Bacillus cereus 172560W]gi|229180445|ref|ZP_04307788.1∥gnl|WGS:NZ_ACLV01|bcere0005_37900[229180445];Leucine dehydrogenase [Bacillus cereus MM3]gi|2291748411ref|ZP_04302361.1∥gnl|WGS:NZ_ACLW01|bcere0006_39250[229174841];Leucine dehydrogenase [Bacillus cereus AH621]gi|229168909|ref|ZP_04296626.1∥gnl|WGS:NZ_ACLX01|bcere0007_38620[229168909];Leucine dehydrogenase [Bacillus cereus R309803]gi|229163100|ref|ZP_04291056.1∥gnl|WGS:NZ_ACLY01|bcere0009_38690[229163100];Leucine dehydrogenase [Bacillus cereus ATCC 4342]gi|229157746|ref|ZP_04285821.1∥gnl|WGS:NZ_ACLZ01|bcere0010_39270[229157746];Leucine dehydrogenase [Bacillus cereus m1550]gi|229152367|ref|ZP_04280559.1∥gnl|WGS:NZ_ACMA01|bcere0011_39050[229152367];Leucine dehydrogenase [Bacillus cereus BDRD-ST24]gi|229146739|ref|ZP_04275105.1∥gnl|WGS:NZ_ACMB01|bcere0012_38800[229146739];Leucine dehydrogenase [Bacillus cereus BDRD-ST26]gi|229140899|ref|ZP_04269444.1∥gnl|WGS:NZ_ACMC01|bcere0013_39930[229140899];Leucine dehydrogenase [Bacillus cereus BDRD-ST196]gi|229134979|ref|ZP_04263785.1∥gnl|WGS:NZ_ACMD01|bcere0014_38860[229134979];and Leucine dehydrogenase [Bacillus cereus BDRD-Cer4]gi|229129445|ref|ZP_04258416.1∥gnl|WGS:NZ_ACME01|bcere0015_38880[229129445].

Leucine dehydrogenases useful with the process of the present disclosureinclude an L-leucine dehydrogenase from Bacillus cereus such as theenzyme disclosed in e.g., U.S. Pat. No. 5,854,035, which is herebyincorporated by reference herein.

Exemplary leucine dehydrogenases useful with process of the disclosurecan also be obtained from Geobacillus stearothermophilus (e.g.,gi|34014423|dbj|BAC81829.1|; gi∥43145|gb|M22977.1|;giβ4014421|dbj|AB103384.1|). In one embodiment, an L-leucinedehydrogenase from G. stearothermophilus useful with the present processcomprises an amino acid sequence of SEQ ID NO: 26. Further suitableLeuDH enzymes useful with the present process can be engineered bysite-directed mutagenesis and/or directed evolution methods using thepolynucleotide of SEQ ID NO: 25, which encodes the wild-type LeuDH ofSEQ ID NO: 26.

As noted above, the ammonium ion donor in the process mediated by theleucine dehydrogenase can be any suitable ammonium ion donor, whichprovides the NH₃ for formation of the amino acid. Exemplary ammoniumsource includes, among others, various ammonium salts, such as ammoniumhalide (e.g., ammonium chloride), ammonium formate, ammonium sulfate,ammonium phosphate, ammonium nitrate, ammonium tartrate, and ammoniumacetate. In particular, the process for forming L-tert-leucine withleucine dehydrogenase can use ammonium chloride as the ammonium donor.

In the process for formation of L-tert-leucine, the ketoreductase of thecofactor regenerating system can be any suitable ketoreductase capableof forming reduced cofactor NADH and/or NADPH utilizing the oxidizationof the alcohol to the corresponding carbonyl compound to drive thereaction towards reduced cofactor formation. As described herein, theketoreductase can be a wild type ketoreductase, or an engineeredketoreductase. The engineered ketoreductase can have an improved enzymeproperty, such as improvements in enzymatic activity, thermostability,solvent stability, pH stability, inhibitor resistance, or cofactorpreference. Ketoreductases found in or derived from any number oforganisms, as discussed above, can be used in conjunction with theleucine dehydrogenase.

In some embodiments, the ketoreductase is an engineered enzyme derivedfrom Candida magnoliae, Lactobacillus kefir, Lactobacillus brevis,Lactobacillus minor, Saccharomyces cerevisiae or Novosphingobiumaromaticivorans, as discussed above. Exemplary engineered ketoreductasesuseful with the leucine dehydrogenase in forming L-tert-leucine cancomprise an amino acid sequence selected from the group consisting ofSEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24.

In the leucine dehydrogenase mediated process, the substrate alcohol forthe ketoreductase can comprise any alcohol that is recognized andconverted by the ketoreductase to the corresponding carbonyl compound.The alcohol can be a primary alcohol or a secondary alcohol. Anexemplary primary alcohol recognized by ketoreductases is ethanol. Insome embodiments, the alcohol is a suitable secondary alcohol, includinglower secondary alkanols and aryl-alkyl carbinols, as noted above.Exemplary secondary alcohol for use in the cofactor regenerating systemwith the leucine dehydrogenase includes, by way of example and notlimitation, include isopropanol, 2-butanol, 3-methyl-2-butanol,2-pentanol, 3-pentanol, 3,3-dimethyl-2-butanol, and the like,particularly isopropanol.

The alcohol, particularly a secondary alcohol, such as isopropanol, canbe present at about 1% to 60% v/v, about 1% to 50% v/v, about 1% to 40%v/v, about 1% to 30% v/v, about 1% to 20% v/v, or about 1% to 10% v/v ofthe reaction medium. In some embodiments, the alcohol can be present atabout 10% to 60% v/v, about 10% to 50% v/v, about 10% to 40% v/v, about10% to 30% v/v, or about 10% to 20% v/v of the reaction medium. In someembodiments, the alcohol can be present at about 20% to 60% v/v, about20% to 50% v/v, about 20% to 40% v/v, or about 20% to 30% v/v of thereaction medium. In some embodiments, the process with the leucinedehydrogenase has about 5% to 20% isopropanol, particularly 5% to 15%isopropanol. An exemplary amount of isopropanol is about 8% to 12% ofthe reaction medium v/v. In some embodiments, the secondary alcohol ispresent in at least 1.5 fold stoichiometric excess of substrate.

Generally, the process with the leucine dehydrogenase can be carried outat a pH of about 11 or below, usually in the range of from about 8.0 toabout 11. In some embodiments, the process is carried out at a pH ofabout 9.0 or below, usually in the range of from about 8.5 to about 9.0.In some embodiments, the process for forming L-tert-leucine with leucinedehydrogenase, can be carried out at a pH of about 9 to about 11,particularly at about pH 9 to 10, more particularly at about pH 9.5. Insome embodiments, the process may be carried out a neutral pH, i.e.,about 7.0. It is to be understood that the pH of the reaction medium forthe formation of L-tert-leucine can be determined based on theactivities of the leucine dehydrogenase and ketoreductase at differentpHs.

In some embodiments, the process described herein can be carried out ata temperature in the range of from about 15° C. to about 75° C. In someembodiments, the reaction is carried out at a temperature in the rangeof from about 20° C. to about 55° C. In some embodiments, it is carriedout at a temperature in the range of from about 20° C. to about 45° C.In some embodiments, the process is carried out at a temperature ofabout 35° C. to about 45° C. The reaction may also be carried out underambient conditions.

In view of the foregoing, a process for producing(S)-2-amino-3,3-dimethylbutanoic acid (also referred to herein as“L-tert-leucine”), can comprise: contacting 3,3-dimethyl-2-oxobutanoicacid (also referred to herein as “trimethyl pyruvic acid”, “TMP”, or“trimethyl pyruvate”) with a reaction medium comprising a leucinedehydrogenase, an ammonium ion donor, NAD+/NADH or NADP+/NADPH, and acofactor recycling system comprising a ketoreductase and a lowersecondary alcohol, under conditions where the 3,3-dimethyl-2-oxobutanoicacid is converted to (S)-2-amino-3,3-dimethylbutanoic acid, wherein the3,3-dimethyl-2-oxobutanoic acid is at about 75 g/L to 125 g/L, thecofactor is at about 0.30 g/L to 0.70 g/L, and the leucine dehydrogenaseand ketoreductase are each independently at about 0.5 to about 1.0 g/L.

The process for forming L-tert-leucine with a leucine dehydrogenase canbe carried out with whole cells or in a cell free system. In someembodiments, the leucine dehydrogenase can be present in whole cellswhile the ketoreductase is present in a cell free system. Conversely, insome embodiments, the leucine dehydrogenase can be present in cell freesystem while the ketoreductase is present in whole cells. In someembodiments, the whole process is carried out in a cell free system,where the leucine dehydrogenase and the ketoreductase is present a crudeextract or in isolated form. In some embodiments, the leucinedehydrogenase and ketoreductase is provided in substantially purifiedform.

In some embodiments, the process for forming L-tert-leucine of thepresent disclosure can be carried out wherein the leucine dehydrogenasepolypeptide and/or the ketoreductase polypeptide is immobilized on asurface, for example wherein the enzyme is linked to the surface of asolid-phase particle (e.g., beads) or resin that is present in thesolution or otherwise is contacted by the solution (e.g., a flow-throughsystem in a column). Methods for linking (covalently or non-covalently)enzymes to solid-phase supports or particles (e.g., porous or non-porousbeads, resins, or solid supports) such that they retain activity for usein industrial bioreactors are known in the art (see e.g., Hermanson, G.T., Bioconiugate Techniques, Second Edition, Academic Press; (2008), and“Bioconjugation Protocols: Strategies and Methods,” in Methods inMolecular Biology, C. M. Niemeyer ed., Humana Press (2004); Mateo etal., “Epoxy sepabeads: a novel epoxy support for stabilization ofindustrial enzymes via very intense multipoint covalent attachment,”Biotechnol Prog. 18(3):629-34 (2002); the disclosures of which areincorporated herein by reference). Useful solid supports can be in theform of beads, spheres, particles, granules, a gel, a membrane or asurface, can be porous or non-porous, can have swelling or non-swellingcharacteristics, can be composed of organic polymers, such aspolystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof, or can be composed of inorganic solids, such as glass, silica,controlled pore glass (CPG), reverse phase silica or metal, such as goldor platinum. A variety of useful solid supports for the immobilizationof enzymes are commercially available (e.g., SEPABEADS resins; availablefrom Sigma-Aldrich, USA).

In some embodiments of the process using immobilized enzymes, theimmobilized LeuDH and KRED polypeptides can be on different particles,which can be mixed in the reaction chamber, or in some embodiments, bothenzymes can be immobilized on the same particles. In some embodiments,the methods using immobilized polypeptides can be carried out whereinthe method further comprises a step of isolating or separating theimmobilized enzymes from the reaction solution containing the product sothat the enzymes can be recycled. In continuous flow-throughembodiments, the particles comprising the immobilized LeuDH and/or KREDare maintained in a reaction chamber or column and the reacting solutionflows through at a rate (and under suitable conditions) to allow forcomplete conversion of the substrate the reaction solution to productwhereupon it exits the reaction chamber or column.

As noted above, to facilitate the reaction toward formation of product,the process of can further comprise removing from the reaction mediumthe carbonyl product formed from the alcohol used as the substrate tothe ketoreductase. In the exemplary embodiments using isopropanol as thealcohol, the corresponding product acetone can be removed by spargingthe reaction medium with an inert gas, such as nitrogen gas, or bylowering the pressure of the reaction medium and trapping the volatileacetone in a condenser.

As will be apparent to the skilled artisan, the process for formingL-tert-leucine can be varied with respect to amounts of L-leucinedehydrogenase, 3,3-dimethyl-2-oxobutanoic acid, cofactor, ketoreductase,and alcohol for the efficient conversion of the substrate to the product(S)-2-amino-3,3-dimethylbutanoic acid. In some embodiments, at asubstrate loading of at about 75 g/L to 125 g/L, the process is capableof converting at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or about 100% of the substrate tothe corresponding product.

In some embodiments, the present disclosure provides a process forpreparing an N-protected chiral amino acid compound, where the processcomprises: (i) a biocatalytic step for converting a compound of formulaI to a chiral amino acid compound of formula IIa; and (ii) a chemicalstep where the amino acid compound of formula IIa is converted to anN-protected chiral amino acid compound.

The present disclosure contemplates that the biocatalytic step forconverting a compound of formula I to a chiral amino acid compound offormula IIa may be carried out using any of the processes disclosedherein wherein the biocatalytic step comprises contacting a compound offormula I with a reaction medium comprising an amino acid dehydrogenase,an ammonium ion donor, NAD+/NADH or NADP+/NADPH, and a cofactorregenerating system comprising a ketoreductase and a lower secondaryalcohol under suitable conditions where the compound of formula I isconverted to the chiral amino acid compound of formula IIa and the lowersecondary alcohol is converted to a ketone.

The present disclosure contemplates that the chemical step where theamino acid compound of formula IIa is converted to an N-protected chiralamino acid compound is carried out by contacting the amino acid compoundof formula IIa (produced in the biocatalytic step) with a N-protectinggroup reagent under conditions where the N-protecting group reacts withthe amine group nitrogen of the compound of formula IIa.

Examples of such N-protecting groups useful in the processes of thedisclosure include the formyl group, the trityl group, the methoxytritylgroup, the tosyl group, the phthalimido group, the acetyl group, thetrichloroacetyl group, the chloroacetyl, bromoacetyl, and iodoacetylgroups, benzyloxycarbonyl (Cbz), methoxycarbonyl (MOC),9-fluorenylmethoxycarbonyl (FMOC), 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), trihaloacetyl, benzyl, benzoyl, andnitrophenylacetyl and the like. Further examples of protecting groupsuseful with the embodiments of the present disclosure can be found in P.G. M. Wuts and T. W. Greene, “Greene's Protective Groups in OrganicSynthesis—Fourth Edition,” John Wiley and Sons, New York, N.Y., 2007,Chapter 7 (“Greene”).

In one embodiment, the compound of formula I is trimethylpyruvic acid(TMP) and the chiral amino acid compound of formula IIa isL-tert-leucine. According to the process of the present disclosure anN-protected L-tert-leucine (e.g., an L-tert-leucine with its amine groupnitrogen protected) can be prepared with e.g., a t-butoxycarbonyl (BOC),benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl (FMOC), ormethoxycarbonyl (MOC) protecting group. Processes for preparing the BOCand MOC protected L-tert-Leucine are described in Examples 1-4.

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

EXAMPLES Example 1: Biocatalytic Process for Preparation ofEnantiomerically Pure L-Tert-Leucine from Trimethylpyruvic Acid (TMP)

This Example illustrates a biocatalytic process for production ofenantiomerically pure L-tert-leucine from trimethylpyruvic acid at a 20g scale using leucine dehydrogenase (LeuDH) in the presence of aketoreductase (KRED) recycling system.

Materials

The materials used and their sources are provided in Table 5. Specificvendors and grades are given for commercially available inputs, althoughit is expected that the source of such reagents would not have an impactupon the reaction.

TABLE 5 Materials List Material Amount Trimethylpyruvic acid 20 g (60%aqueous solution) (33.4 mL) Water 118.6 mL Ammonium hydroxide 19 mL(28-30% aqueous solution) Ammonium chloride 9.0 g Isopropanol (min99.7%) 20 mL β - NAD⁺ 100 mg Leucine dehydrogenase 150 mg (LeuDH) of SEQID NO: 26 Ketoreductase (KRED) of 150 mg SEQ ID NO: 18Summary of Reaction Conditions

A summary of the reaction conditions used in the biocatalytic processare shown below in Table 6.

TABLE 6 Reactant parameters and conditions for biocatalytic processTrimethylpyruvic acid (TMP) 100 g/L Ammonium chloride 1.1 equiv pH 9.0IPA 10% β - NAD⁺ 0.50 g/L LeuDH of SEQ ID NO: 26 0.75 g/L KRED of SEQ IDNO: 18 0.75 g/L Temperature 40° C.Reaction Protocol

A 250 mL 2-neck round bottom flask was equipped with pH probe and amagnetic stirrer. The pH probe also served as an internal thermometer.The flask was charged sequentially with 100 mL water and 20.0 g (33.4mL) of trimethylpyruvic acid. The resulting aqueous solution was ˜pH 1.1(measured at 23° C.) and was stirred for 10 minutes at 400 rpm at roomtemperature to obtain homogeneity. The solution was cooled to 15° C.using ice water bath. Ammonium hydroxide was added in portions toneutralize the acid and attain a pH between 7 and 7.5, maintaining thetemperature below 20° C. during addition. Approximately 13 mL ofammonium hydroxide (28-30% aqueous solution) adjusted the pH to 7.34 at21° C. Ammonium chloride (1.1 equiv, 9.0 g) was added and stirred untila clear solution was obtained. After addition of ammonium chloride,reaction mixture was allowed to reach room temperature and the pH atroom temperature approximately 7.0. The reaction mixture was readjustedto pH 9.0 by adding ammonium hydroxide in portions, maintaining thetemperature below 25° C. during addition. Approximately 6 mL of ammoniumhydroxide (28-30% aqueous solution) adjusted the pH to 9.03 at 22° C.The pH was found to have a significant impact on the rate of reactionReactions did not proceed to complete conversion in 24 h when NaOH wasused to adjust pH to pH 9.5 or above. 20 mL isopropanol (IPA) was addedto the reaction mixture. The pH dropped to 8.95 upon addition of IPA,but no further re-adjustment to pH 9.0 was carried out. NAD (100 mg) wascharged to the stirred mixture as a powder and 18.6 mL of water as addedto the reaction mixture to adjust the final volume to 200 mL. Theleucine dehydrogenase (LeuDH) of SEQ ID NO: 26 (150 mg) and theengineered ketoreductase (KRED) of SEQ ID NO: 18 (150 mg) biocatalystswere charged to the stirred mixture as powders. The reaction mixture washeated to 40° C. (internal temperature) stirring at 600 rpm. The finaltemperature was reached within 30 min. The reaction course was followedby taking samples periodically out of the reaction mixture and analyzingas described below. For the purpose of monitoring the reaction, t=0 wasthe time at which the KRED was added into the reaction mixture. Afterin-process analyses indicated maximum conversion, the reaction wascooled to room temperature (in this Example, the reaction was stirredfor a total time of 24 h). As shown by the reaction profile based on thein-process analyses (described further below) provided in Table 7 below,the biocatalytic reaction achieved nearly 88% conversion of TMPsubstrate to L-tert-leucine product in only 7.5 hours, and complete(i.e., 99.9%) conversion of substrate to L-tert-leucine product in 24hours.

TABLE 7 Reaction profile Time (h) % Conversion 0 0 1.5 53.7 3 64.0 4.575.6 6 83.7 7.5 87.9 22 99.5 24 99.9

White turbidity was observed as reaction progressed and the pH of thereaction mixture after complete conversion was 8.83 at room temperature.A sample (10 mL) of the reaction mixture was taken and heated at 40° C.for an additional 24 h. No increase in impurities was observed by GCanalysis.

L-Tert Leucine Product Isolation and Work-Up

The reaction mixture was adjusted to pH 4.5 using 6.0 M HCl solutionunder controlled exothermic conditions. Celite (3.0 g) was added at roomtemperature and stirred to obtain homogeneity. The mixture was filteredthrough a sintered glass funnel and the filter cake washed with 10 mL ofwater. The combined aqueous solution was concentrated to about one-thirdthe original volume. Precipitation of a white solid was observed duringconcentration. The suspension was cooled in an ice bath for a few hoursand the solid was filtered, washed with 20 mL of a 1:1 mixture ofisopropanol:water followed by 20 mL of acetonitrile. A repeat washingwith 1:1 isopropanol:water (50 mL) and acetonitrile (50 mL) afforded 3.0g (approx. yield=50%) of white solid. The solid was identified as thedesired product, L-tert-leucine, with about 90% purity when comparedwith a standard sample of L-tert-leucine (peak area by HPLC-ELSD).

Analytical Methods

Analytical methods used in the biocatalytic process and described belowinclude: Method 1, a GC analysis method used to monitor % conversion oftrimethylpyruvic acid substrate to L-tert-leucine product; Method 2, anHPLC-UV-ELSD method to monitor trimethylpyruvic acid substrate to andL-tert-leucine product in the reaction; and Method 3, an HPLC method todetermine the enantiomeric excess of L-tert-leucine formed during thecourse of the reaction. Results obtained by the Method 1 were verifiedusing Method 2.

Method 1: GC Analysis Method for Monitoring % Conversion

Sample Preparation:

In a 1.5 mL glass vial, samples from the reaction mixture (5 μL) werediluted in 1.0 mL of acetonitrile containing 10 μL of pyridine. Methylchloroformate (10 μL) was added and the mixture was agitated for a fewseconds. The exothermic reaction accompanied by release of CO₂ was leftstanding until the precipitate settled. A sample (300 μL) from thesupernatant of the derivatized reaction sample was taken and analyzed bygas chromatography (GC). A summary of the analytical parameters andconditions are provided in Table 8 below.

TABLE 8 Instrument Agilent 6890N series Column: HP-5 (30.0 μm × 320 μm ×0.25 μm nominal) Temperature profile: 90° C. (hold for 2.8 min) thenramp to 210° C. @ 20° C./min Pressure: 12 psi (constant) Inlet: 250° C.Split ratio 20:1 Detector FID, 250° C. Run time 8.8 min Retention timesTrimethylpyruvic acid methyl ester (Peak 1): 2.50 min DerivatizedL-tert-leucine (Peak 2): 6.6 min

The % conversion based on consumption of trimethylpyruvic acid wascalculated as follows:

${\%\mspace{14mu}{Conversion}} = {\frac{\left( {{Area}\mspace{14mu}{of}\mspace{14mu}{Peak}\mspace{14mu} 1} \right)_{t = 0} - \left( {{Area}\mspace{14mu}{of}\mspace{14mu}{Peak}\mspace{14mu} 1} \right)_{t = x}}{\left( {{Area}\mspace{14mu}{of}\mspace{14mu}{Peak}\mspace{14mu} 1} \right)_{t = 0}} \times 100}$Method 2: HPLC-UV-ELSD Analysis Method for Monitoring % Conversion

HPLC Sample Preparation:

20 μL of the reaction mixture was taken and added to 980 μL of mobilephase A. Injection is neat into the HPLC equilibrated and set upaccording to the analytical parameters and conditions are provided inTable 9 below.

TABLE 9 Instrument Agilent HPLC 1200 series Column Mightysil RP18 GP,250 × 4.6 mm, 5 μm (1 × Aqua R18 guard column before & after analyticalcolumn). Mobile Phase A: 76% 20 mM NH₄OAc, pH 4.87 + 0.3 mMdodecyltriethylammonium phosphate [Regis Cat No: 404021] B: 24% MeCNFlow rate 1.0 mL/min Run Time 15 min ELSD detector temperature 35° C.Column temperature 29° C. Injection volume 10 μL Gain 3 UV Wavelength230 nm Retention Times Peak 1 (L-tert-leucine): 2.725 min Peak 2 (TMP):13.587 min

The % conversion is calculated as follows:

${\%\mspace{14mu}{Conversion}} = {\frac{\left( {{Area}\mspace{14mu}{of}\mspace{14mu}{Peak}\mspace{14mu} 1} \right)}{\left\lbrack {\left( {{Area}\mspace{14mu}{of}\mspace{14mu}{Peak}\mspace{14mu} 1} \right) + \left( {{Area}\mspace{14mu}{of}\mspace{14mu}{Peak}\mspace{14mu} 2 \times {Response}\mspace{14mu}{factor}} \right)} \right\rbrack} \times 100}$where the Response Factor is calculated as follows:

${{Response}\mspace{14mu}{Factor}} = \frac{\left( {{{Area}\mspace{14mu}{of}\mspace{14mu}{Peak}\mspace{14mu} 1\mspace{14mu}{at}\mspace{14mu}{time}} = x} \right)}{\left\lbrack {\left( {{{Area}\mspace{14mu}{of}\mspace{14mu}{Peak}\mspace{14mu} 2\mspace{14mu}{at}\mspace{14mu}{time}} = 0} \right) - \left( {{{Area}\mspace{14mu}{of}\mspace{14mu}{Peak}\mspace{14mu} 2\mspace{14mu}{at}\mspace{14mu}{time}} = x} \right)} \right\rbrack}$Method 3: HPLC Analysis of Enantiomeric Purity

HPLC Sample Preparation:

10 μL from the reaction mixture was taken and added 990 μL of mobilephase in a 2 mL HPLC glass vial. Injection is neat into the HPLCequilibrated and set up according to the analytical parameters andconditions are provided in Table 10 below.

TABLE 10 Instrument Agilent HPLC 1200 series Column Phenomenex Chirex4.6 × 150 mm (5 μm) Mobile Phase 85% 3 mM CuSO₄, 15% MeOH Flow Rate 1.00mL/min Detection Wavelength 254.0 nm Detector Temperature 25° C.Injection Volume 10 μL Run time 20.0 min Retention Times: Peak 1(L-tert-leucine): 11.319 min Peak 2 (D-tert-leucine): 19.560

Example 2: 800 Gram-Scale Biocatalytic Process for Preparation ofEnantiomerically Pure L-Tert-Leucine from Trimethylpyruvic Acid (TMP)

This Example illustrates a scaled up version of the biocatalytic processfor production of enantiomerically pure L-tert-leucine fromtrimethylpyruvic acid as described in Example 1 to near-kilogram scale.Generally, the reaction protocol followed the same general schemedescribed in Example 1, only at a larger scale.

The materials used and their sources are provided in Table 11.

TABLE 11 Material Quantity Trimethyl pyruvate 800 g (TMP) Ammoniumformate 128 g Ammonia (25% w/w) 750 mL Isopropanol (IPA) 800 mL NAD 4 gLeuDH 12 g (SEQ ID NO: 26) KRED 12 g (SEQ ID NO: 18) Deionized water 5.5L (DIW) Acetone 11 L Celite 545 250 gReaction Protocol

A 10 L two neck round bottom flask in a water bath was charged 800 g TMPsubstrate in ˜5 L water. Overhead stirrer (equipped with flat two-bladepaddle; 8 cm diameter) was started and water bath heated to 40.0° C. Theammonia was added in portions until all TMP was dissolved (approx.temperature increase of 4° C.). The flask was then charged ammoniumformate and IPA. The pH was adjusted with ˜170 mL ammonia to pH 9.0±0.1at 40.0° C.±0.5° C. The solution was charged with 12 g of KRED of SEQ IDNO: 18, 12 g of LeuDH of SEQ ID NO: 26, and 4 g of NAD in 500 mL water.The reaction volume was adjusted to 8 L with water as necessary. After18 h the reaction was complete (as determined by Method 1 analysis ofExample 1).

Workup Procedure

Charged 250 g Celite 545 and stir for about 15 min then filtered offCelite with a 5 L P4 fritted funnel. Distilled off ammonia, acetone, IPAand water at 50° C. under reduced pressure up to 60 mbar, until a vesselvolume of ˜2.5 L is achieved. Charged residue in a 15 L bucket with 10 Lcold acetone (4 vols) and mixed for 5 min. Closed bucket was storedovernight at 4° C. Precipitate was filtered off and washed two timeswith 500 mL cold acetone. Filter cake was dried at 50° C. under reducedpressure yielding 624 g L-tert-Leucine as a beige powder having >95%e.e.

The crude beige powder from this workup procedure could be immediatelyused in the reaction with the N-protecting groups MOC or BOC asdescribed in Example 3, to produce the N-protected version ofL-tert-leucine.

Example 3: Preparation of MOC-Protected L-Tert-Leucine

This Example illustrates a process for production of a MOC-protectedL-tert-leucine compound made using L-tert-leucine preparedbiocatalytically using LeuDH and an engineered KRED recycling system asdescribed in Examples 1 or 2.

Materials used in the process are shown in Table 12.

TABLE 12 Material Quantity L-tert-Leucine 10.0 g Deionized water (DIW)80 mL Methyl chloroformate (MOC-Cl) 14.4 g NaOH, 50% wt/wt aqueous 21.4g Hydrochloric acid, 12N 12 mL Isopropyl acetate (iPrOAc) 8.0 mL Heptane(Hept) 125 mL Ethyl acetate (EtOAc) 120 mL NaCl, saturated aqueous 10 mLEquipment and Reaction Protocol

A 300 mL round-bottomed flask fitted with a mechanical stirrer,thermocouple, and graduated addition funnel was used for the reaction,extractive workup. Temperature was controlled with a water/ice bath forbelow ambient temperature and a calibrated pH probe and meter used atcertain stages. For isolation the vessel was fitted with a short-pathdistillation head.

The reaction vessel was charged with 10.0 g crude L-tert-Leucine (e.g.,made as in Example 2) and 70 mL water, and the stirrer set at 150 rpm.21.4 g of NaOH added to the addition funnel and fed dropwise into thereaction over 5 min (temperature remains <35 C) resulting in astraw-colored solution. The addition funnel was rinsed with 10 mL waterand then charged with 14.4 g MOC-Cl, 14.4 g. The MOC-Cl was fed into thereaction dropwise over 30-40 min (temperature does not exceed 50° C.).HPLC was used to monitor the reaction for completion of conversion.

Workup Procedure

Upon complete conversion a pH probe was inserted and the flask allowedto cool <25 C. Approximately 12 mL of 12 N HCl was fed drop wise usingthe addition funnel until the pH was ˜2 while keeping thetemperature<30° C. The reaction was charged with 60 mL ethyl acetatatewhich was mixed for 2 min and then layers allowed to separate (emulsionswere filtered through an “M” sintered frit). The upper organic layer wasremoved and stored. The pH was re-adjusted to ˜pH 3 and the extractionwas repeated two more time with 40 mL then 20 mL of ethyl acetate. Theextracted upper organic layers were combined and charged the 10 mLsaturated NaCl with mixing for 2 min followed by layer separation. Theorganic layers (˜130 mL) were charged to a reaction vessel fitted with adistillation kit. Heat was applied to distill solvent (with 200 rpmstirring) until a vessel volume of 80 mL is achieved. Continuedistillation with heptane fed dropwise to maintain ˜80 mL vessel volume.Continue distillation and feed until the pot temperature is ˜98.2 C.Cool to 80° C. and feed iPrOAc, 8 mL. Cool to 65° C. and charge productseed crystals, 0.1 g. Slowly cool to ambient and then to 5 C. Collectproduct by filtration on an “M” sintered glass funnel; wash through withrecycled liquors until all solid is collected. Wash solid with heptane,15 mL, and suction dry. Vacuum dry to constant weight at 40° C.

Analytical Methods

HPLC was utilized for in-process analysis of % conversion and finalproduct quality assay. H-NMR was utilized only for final product qualityassay for general purity and as a check for residual solvents.

Sample Preparation:

During the reaction, with mixer on, a 200 μL aliquot is withdrawn viapipette and diluted into 3000 μL of acetonitrile/water (50:50). Thissample is shaken by hand to ensure homogeneity. The sample is furtherdiluted 1:2 prior to injection.

The analytical instrumentation and relevant parameters are shown inTable 13.

TABLE 13 Instrument Agilent 1100 Column TOSOH Bioscience, #19533, TSKGelAmide80 (4.6 mm × 10 cm, 5 μm) Column temp 30° C.  Detection 205 nm Flowrate    1.2 mL/min Mobile phase C = water; D = acetonitrile Timetable:Time (min) % C % D 0 0.2 99.8 2.00 1 99.0 10.00 60 40 12.00 0.2 99.8Injection volume  10 μL Sample Concentration   ~5 mg/mL Retention Timestert-leucine: 7.8 min MOC-tert-leucine: 1.7 min

Example 4: Preparation of BOC-Protected L-Tert-Leucine

This Example illustrates a process for production of a BOC-protectedL-tert-leucine compound made using L-tert-leucine preparedbiocatalytically using a ketoreductase (KRED) recycling system asdescribed in Examples 1 or 2.

Materials used in the process are shown in Table 14.

TABLE 14 Material Quantity L-tert-Leucine 10 g Deionized water (DIW) 70mL Triethylamine (TEA) 7.71 g di-tert-butyl dicarbonate (BOC₂O) 20.8 gNaOH, 50% wt/wt aqueous 9.15 g Tetrahydrofuran (THF) 30 mL Heptane 285mL Ethyl acetate 145 mL NaCl, saturated aqueous 10 mLEquipment and Reaction Protocol

Reaction Vessel:

A 300 mL round-bottomed flask fitted with a mechanical stirrer,thermocouple, and graduated addition funnel was used for the reactionand extractive workup. Temperature was controlled with a water/ice bathfor below ambient temperature. A calibrated pH probe and meter was usedat certain stages.

Isolation Vessel:

A 500 mL round-bottomed flask fitted with a mechanical stirrer,thermocouple with temperature controller and heating mantle, graduatedaddition funnel, and distillation kit was used for the solvent exchangeand crystallization.

Reaction vessel was charged 10.0 g crude L-tert-Leucine product (as madein Example 2), and 70 mL to the reaction vessel and set stirrer at 150rpm. Solution was a suspension. Charged TEA, 7.71 g, over 5 min so thatT remains <25° C., which resulted in a straw-colored solution. Chargedreaction vessel with NaOH, 4.57 g, over 2 min, resulting in an opaquesolution. Prepared a solution of BOC₂O, 20.8 g., in THF, 30 mL, and feddropwise to reaction vessel over 30-40 min, without allowing temperatureto exceed 32° C. The reaction was monitored for complete conversion byHPLC as described in Example 3, except that retention time forBOC-tert-leucine was 1.5 min.

Workup Procedure

Checked pH and adjusted with 50% NaOH to pH>9.2. Charged vessel withheptanes (30 mL) with mixing. Upper organic layer was removed and a pHprobe and flask cooled to <25° C. before charging with ˜32 mL 3 N HCldropwise (pH˜3.5) and keeping temperature<30° C. Remove cooling andcharged with ethyl acetate (50 mL) followed by mixing for 2 min andremoval of upper organic layer. pH was readjusted to ˜3.5, if necessary,and the vessel charged with ethyl acetate (70 mL) and heptane (30 mL).Upper organic layer removed and stored. Charged again with ethyl acetate(25 mL) and heptane (25 mL). Upper organic layer removed and stored. Allorganic phases were combined and filtered through an “M” sintered fritif an emulsion were present.

The combined organic phases were charged with sat'd NaCl, 10 mL, mixedfor 2 min and allowed to separate. Lower layer was removed (˜235 mL) andcharged to a crystallizer fitted with a distillation kit. Heat wasapplied to distill solvent until a vessel volume of 100 mL is achieved.Continue distillation and begin feeding heptanes dropwise so as tomaintain ˜100 mL vessel volume. Continue distillation and feed until thepot temperature˜98° C. Cool to 5° C. and collect product by filtrationon an “M” sintered glass funnel. Wash through with recycled liquorsuntil all solid is collected. Wash solid with pre-cooled (5° C.)heptane, 15 mL. Vacuum dry to constant weight at 40° C.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. A process for converting a compound of formula Iwhich is a substrate for an amino acid dehydrogenase to a chiral aminoacid of formula IIa:

comprising contacting the compound of formula I with a reaction mediumcomprising an amino acid dehydrogenase, an ammonium ion donor selectedfrom ammonia or an ammonium salt, a cofactor selected from NAD⁺/NADH orNADP⁺/NADPH, and a cofactor regenerating system comprising an engineeredketoreductase encoded by an engineered polynucleotide having at least90% sequence identity to SEQ ID NO: 17, which has less than 5% of theactivity of an amino acid dehydrogenase, with compound of formula I anda lower secondary alcohol, under conditions where the compound offormula I is converted to the chiral amino acid of formula IIa and thelower secondary alcohol is converted to a ketone, and wherein R is asubstituted or unsubstituted —(C₁-C₁₀)alkyl, —(C₂-C₆)alkenyl,—(C₂-C₆)alkynyl, —(C₃-C₈)cycloalkyl, heterocycloalkyl, aryl, orheteroaryl.
 2. The process of claim 1, wherein the lower secondaryalcohol is isopropanol and the ketone is acetone.
 3. The process ofclaim 1, further comprising the step of removing the lower secondaryalcohol formed from the lower alkyl ketone from the reaction medium. 4.The process of claim 1, wherein the reaction medium is at a pH of about8.5 to about
 10. 5. The process of claim 4, wherein the reaction mediumis at a pH of about 8.5 to about 9.0.
 6. The process of claim 1, whereinthe reaction medium is at a temperature of about 25° C. to about 45° C.7. The process of claim 6, wherein the reaction medium is at atemperature of about 35° C. to about 40° C.
 8. The process of claim 1,wherein the secondary alcohol is present in at least 1.5 foldstoichiometric excess of substrate.
 9. The process of claim 2, whereinthe ketoreductase is capable of recycling cofactor by convertingisopropanol (IPA) to acetone in a reaction medium of 3 to 20% IPA at apH of about 9.0 to 10.5 with an activity at least 2.0-fold greater thana reference ketoreductase encoded by the polynucleotide of SEQ ID NO:1.10. The process of claim 1, wherein the activity of the ketoreductasewith compound of formula I is less than 2%, 1%, 0.5%, 0.1%, 0.05%, or0.01% of the activity of the amino acid dehydrogenase with the compoundof formula I, or optionally no activity with the compound of formula I.11. The process of claim 1, wherein the compound of formula IIa is3,3-dimethyl-2-oxobutanoic acid and the product of formula I is(S)-2-amino-3,3-dimethylbutanoic acid and the amino acid dehydrogenasecomprises a L-leucine dehydrogenase.
 12. The process of claim 1, whereinthe ketoreductase comprises an amino acid sequence encoded by anengineered polynucleotide selected from SEQ ID NO:
 17. 13. The processof claim 11, wherein the leucine dehydrogenase comprises an amino acidsequence encoded by an engineered polynucleotide comprising the sequenceof SEQ ID NO:
 25. 14. The process of claim 1, wherein the amino aciddehydrogenase comprises an amino acid sequence encoded by an engineeredpolynucleotide comprising the sequence of SEQ ID NO:
 25. 15. The processof claim 1 which is carried out in a cell free system.
 16. The processof claim 1, wherein the amino acid dehydrogenase is present as a crudeextract.
 17. The process of claim 1, wherein the amino aciddehydrogenase is substantially purified.